The University of Washington 
Department of Chemistry 



A Biochemical Study of Pacific Coast Salmon 
with Particular Reference to the Formation 
of Indol and Skatol During Decomposition 



by 



RAY WILLIAM GLOUGH 
II 



A Thesis Submitted in Partial Fulfilment of the Requirements for 
the Degree of Doctor of Philosophy 



Seattle 

University Press, University of Washington 

1922 



1 



LIBRARY OF CONGRESS 

DECEIVED 

MAY 9 1923 

DOCUMENTS U:\iiS\ON 



A Biochemical Study of Pacific Coast Salmon With Particular 
\J> Reference to the Formation of Indol and Skatol 

During Decomposition 1 



Ray William Clough 2 
University of Washington 



1. The salmon canning industry 196 

2. Examination of commercial canned salmon 200 

A. Systematic method 200 

3. Chemical composition of fish flesh 204 

4. Decomposition of fish flesh 209 

5. Development of a method for detecting decomposition 

by means of indol and skatol i 214 

A. Selection of suitable color tests 214 

B. Modification of the selected color tests 217 

C. Distillation of indol from salmon 219 

D. Extraction of indol from the distillate 221 

E. Evaporation of the ether extract 222 

F. Color tests en the water test solution 223 

G. The method as finally developed 224 

6. Experimental work on the fivp species of salmon in 

different stages of decomposition 225 

A. General outline of the method employed ' 225 

B. Experimental work on king salmon 229 

C. Experimental work on pink salmon 230 

D. Experimental work on sockeye salmon 235 

E. Experimental work on coho salmon 235 

F. Experimental work on chum salmon 240 

G. Discussion of the results 246 

7. Formation of indol by various means 253 

A. Indol formation by bacteria 253 

B. Formation of indol by scorching proteins 256 

C. Formation of indol during the processing of salmon 257 

D. Effect of exhaust on the indol content of canned salmon 258 

8. Other decomposition changes 1 259 

A. Volatile nitrogen as a measure of decomposition...' 259 

B. Increase in free fatty acids as a measure of 

decomposition in salmon 263 

C. Formation of a substance having a biting taste 265 

9. Summary 265 

10. Bibliography 268 

1 This investigation was made at the University of Washington in connection with other 
problems affecting the salmon canning industry. 

2The writer is deeply indebted to Dean C. W. Johnson of the University of Washington, 
and to Dr. E. D. Clark, Dr. C. R. Fellers, Mr. O. E. Shostrom and Miss M. G. Haslam of 
the National Canners Association, for advice, encouragement and help during the course of the 
investigation. 

(195) 



196 Publ. Puget Sound Biol. Sta. Vol. 3, No. 67 

1. The; salmon canning industry 

The value and importance of the salmon canning industry will 
be realized from a study of the following brief table summarizing 
the number of cases of salmon packed since the inception of salmon 
canning on the Pacific Coast in 1864. This table gives the total cases 
packed for the whole Pacific Coast, including British Columbia, at:d, 
for the sake of brevity, is given in ten-year periods, with the exception 
of the first period which covers only eight years. 

Table; 1. Volume of canned salmon since the inception of the 

industry. 

Period Cases 3 

1864-1871 504,000 

1872-1881 5,013,861 

1882-1891 11,709,915 

1892-1901 26,864,515 

1902-1911 43,244,258 

1912-1921 73,377,223 



Total 1864-1921 160,713,772 



In addition to the salmon which are canned, millions of salm.on 
are preserved each year in various other ways, such as drying, salt- 
ing and freezing. However, more are canned than are preserved 
in all other ways combined. 

Five species of salmon are found in the Pacific Ocean, all belong- 
ing to the genus Oncorhynchus. The Atlantic salmon belongs to a 
distinctly different genus, the genus Salmo. The Pacific salmons are 
found along the whole north semicircle of coast from central Califor- 
nia to central Japan, and some of them have been successfully intro- 
duced into the southern hemisphere, particularly in New Zealand. 
Each of the five species is known by its scientific name, and by sev- 
eral common names, as shown in table 2. 

These five species vary greatly in size, ranging from 3 pound 
(1.4 kg) pinks and sockeyes tc 100 pound (45 kg) kings. The average 
weight is about as follows: Pinks 5 pounds (2.3 kg), sockeyes 5 
(2.3 kg), chums 9 (4kg), medium reds 10 (4.5 kg), and kings 30. 
(13.6 kg). They vary somewhat in habit and life history but all are 
alike in one essential particular, — they are anadromous, that is, when 
they reach maturity each species comes surging in from the sea to 
some fresh water stream or lake to spawn, after which practically all 

3Eeduced to a common basis of 4S one pound (454?.) cans to the case. From data 
given by Cobb (1921). 



1922 Clough; on Indol and Skatol in Salmon 197 

Table 2. Names of the five species of salmon. 



Scientific name 


Puget Sound Columbia River 


Alaska 


Other names 


Oncorhynehus 


Sockeye 


Blueback 


Alaska Red 


Quinault 


nerka 






(Sockeye) 


Redfish 


Oncorhynehus 


Spring 


Chinook 


King 


Tyee, 


tschawytscha 






(Chinook) 


Quinnat 


Oncorhynehus 


Coho 


Silver 


Medium Red, 


Silversides 


kisutch 


Silver 




Coho 




Oncorhynehus 


Pink 


Pink 


Pink 


Humpback 


gorbuscha 










Oncorhynehus 


Chum 


Chum 


Chum 


Keta, Dog, 


keta 








Calico 



of them die and thus complete their life history. The fertilized eggs 
after an interval depending upon many factors, hatch, and the young 
fish spend a certain amount of time in fresh water before going to 
their ocean home. 

The salmon industry depends to a great extent upon this spawn- 
ing migration, for it is only at this time that the salmon may be 
caught in quantity, and indeed three of the species are almost never 
caught at any other time. Coming in from the open se?. in immense 
schools they fall an easy prey to various types of nets, to traps and 
even to hook and line. The fish caught in nets or by hook and line 
(trolling) are taken into the fishermen's boats and often are trans- 
ferred to cannery tenders. After reaching the cannery they are 
unloaded either by sluicing with water into elevators or by pitching 
with one-tined forks called pughs. 

A few of the canneries dress or "butcher" the fish by hand but 
in most plants a machine known as the "iron chink" is used. This 
machine removes the head, tail, fins and viscera, and cleans the body 
cavity by means of brushes and jets of water. The salmon are then 
"slimed" by hand or by machine. In this process the blood, slime, 
loose membranes, etc., are removed with knives, spoons, or machines 
consisting of brushes revolving under jets of water. The cleaned fish 
are then cut into slices suitable for the different sized cans, either 
by a rotary hand-cutter or by "gang knives" consisting of revolving 
disks. The cans, already salted to the extent of 34 ounce (7.1 g) 
of dry salt per 1 pound (454 g) can, are filled by hand or machine. 
A recent type of filling machine cuts the fish into slices, salts the 
cans and fills them at the rate of 115 to 125 per minute. Many of 
the flat cans are filled by hand; this is particularly true of the chinook 



198 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

and sockeye salmon. The filled cans are often weighed by hand or 
machine, and are inspected to see that they have no bones or skin 
showing on top. 

After filling, the cans are usually run through an "exhaust box" 
filled with live steam, in which the contents of the cans becomes 
heated and the air is replaced by steam to a certain extent. This 
may be done before the tops are placed on the cans, but in most can- 
neries the top is first lightly clinched on by machinery to prevent 
particles of fish from getting out and water of condensation from 
getting in. In a few canneries the cans are exhausted by air pumps 
in the "vacuum closing machine." The primary object of exhausting 
the cans is to produce a vacuum which will keep the ends concave 
under changing conditions of temperature and altitude. The hot cans 
from the exhaust pass immediately to the "closing machine" (or 
"double seamer") which rolls the tops on very firmly, making the 
cans air tight. The cans, still hot, are placed in trays ("coolers"), 
stacked on a car and the car pushed along a track into a retort for 
cooking with live steam. Pound (454 g) cans of salmon are cooked 
("processed") usually for 90 minutes at 117° C. (242° F.) or over; 
half pound (227 g) cans a few minutes less. The processing has 
several objects; to cook the fish and make it palatable, to soften the 
bones, and to render it sterile by killing all living organisms within 
the can. The pre-heating received in the "exhaust box" materially 
shortens the time necessary for sterilization. 

After processing, the cans are run through a "lye bath" to re- 
move oil and grease and are then thoroughly washed with water and 
set aside to cool. Leaky or defective cans are detected by tapping the 
bulging ends with a spike or metal rod; the difference in sound is a 
sure test for leaks. After cooling, the cans may be lacquered and 
either stacked in piles, or immediately labeled and placed in cases of 
48 cans each. 

Fish are very delicate, easy to injure and very easily decomposed. 
They must be handled as expeditiously as possible and should be in 
the cans within 48 hours after they are drawn from the water. 
There are many opportunities for spoilage between the time of catch- 
ing and of canning. Salmon caught in gill nets soon suffocate, and 
if not promptly taken from the nets will become somewhat soft. They 
may be left in small boats for a day or more before the boat arrives at 
the cannery or at the cannery tender. 

The fish are frequently transferred from one boat to another, to 
the tender and to the cannery by pitching with one-tined forks. Each 



1922 Clough; on Indol and Skatol in Salmon 199 

time they are thus pughed, bacteria are introduced deeply into the 
hitherto sterile flesh, and from these centers of infection the bac- 
teria rapidly penetrate in every direction. 

Salmon caught by seines are hauled into the boats alive but are 
frequently placed in deep holds to such a depth that the bottom fish 
are badly crushed and softened, and spoil very rapidly, particularly 
since the holds are usually warm and unventilated. A long haul to 
the cannery under such conditions results in considerable spoilage. 
A better practice is to place the fish in bins either in the hold or on 
the deck to a depth of only two feet (61 cm). Separating the fish 
in bins reduces the sliding about and bruising. If the fish are placed 
on the deck they should be covered with boards or tarpaulins as a 
protection from the sun. 

Salmon caught by hook and line as in trolling are usually fish 
which are still feeding, that is, they are either not very far advanced 
on the spawning migration or are immature; mature salmon, reaching 
brackish or fresh water, seldom eat anything more, and thus the di- 
gestive tract is usually empty and free from bacteria. Fish caught 
by trolling, with food in them, deteriorate very rapidly; the bellies in 
time becoming so soft that they break through, a condition known as 
"belly-burning." A peculiar thing about such fish is that although 
their appearance may be very poor they may have no odor of decom- 
position, and in frequent chemical tests we found neither indol nor 
skatol. It is probable that the softening and the breaking down of 
the tissues is due not to bacterial decomposition but to the action of 
the enzymes in the digestive juices. Salmon caught by trolling should 
therefore be cleaned at once and if possible placed on ice. 

Salmon caught in traps are brailed alive into boats or scows and 
should reach the canneiy in good condition if the traps are not 
located too far away and if the boats are not delayed by bad weather. 
Even if the fish reach the cannery in first class condition there may 
be a considerable delay due to an over supply or to a break-down in 
the machinery. It is apparent, therefore, that in handling so perish- 
able a commodity as fish there are many opportunities for spoilage, 
and the excellent condition in which the bulk of the canned salmon 
reaches the market speaks well for the care and resourcefulness of 
the average packer. 

All of the conditions and practices which I have mentioned leave 
their mark upon the fish, and this record may be read in the canned 
product by the experienced examiner. 



200 Publ. Puget Sound Biol. Sta. Vol. 3, No. 67 

2. Examination of commercial canned salmon 

After the salmon has been canned it is very likely to be examined 
at least once before reaching the consumer. Brokers may buy a 
parcel of several thousand cases on the reputation of the packer but 
they are more likely to stipulate that the parcel be examined either 
by themselves or by some one in whom they have confidence.. Little 
or no attempt has been made to grade canned salmon as is done with 
butter, cheese and many other foodstuffs. The only grading has been 
by the species and the district where packed. This is very unsatis- 
factory. If grading is ever placed on a scientific basis it will be by 
means of carefully kept records covering all species and districts. 
To obtain these records a systematic method of examination covering 
everything which is significant regarding the workmanship on the 
parcel and regarding the quality and condition of the fish must be 
followed. Partly as the result of the study of the five species of 
salmon, which is described in the experimental part of this thesis, and 
partly as a result of the careful examination of several thousand cans 
of commercially canned salmon, the following systematic method for 
the examination of canned salmon was evolved. 

A. Systematic method 

Description of the parcel. This should include the species of 
salmon, brand or label, packer, cannery, size of can, can mark, case 
mark, number of cases in the parcel, and location of the parcel. 

Sampling. In canned salmon this is unsatisfactory at best. A 
parcel of 1,000 cases, containing 48,000 cans, may represent a cross 
section of the cannery's entire season's output ranging in extreme 
instances from very good to very bad. One case may even represent 
several days' canning. Under these conditions no systematic method 
of sampling can be carried out. Each case should be stamped with 
the date of packing, then if unsatisfactory fish are found a segrega- 
tion can be made. All that can be done in sampling is to attempt to 
get a representative sample by taking one or two cans from a number 
of cases situated in all parts of the parcel. Ninety-six cans are 
usually drawn in this manner in parcels of 1,000 cases, and an in- 
creasingly smaller proportion from increasingly larger parcels. When 
the cases are opened for sampling they should be inspected for swollen 
or rusty cans and for thoroughness of lacquering and labeling. 

Examination of the sample. This may be undertaken from five 
different viewpoints: (1). Bacteriological examination. (2). Work- 
manship in packing; including the vacuum, cleaning, filling, cooking, 



1922 Clough; on Indol and Skatol in Salmon 201 

salt, pugh marks and net weight. (3). Quality of the fish when 
caught ; here considering the oil, amount of liquid, color and "fresh 
water marking" on the skin. (4). Condition of the fish when canned; 
involving odor, texture, reddening, "honeycombing" and turbidity of 
the liquid. (5). Chemical examination. These are considered in 
order below. 

Bacteriological examination. From 24 to 48 cans are examined 
for living organisms. The cans are carefully cleaned and a gas flame 
is directed upon the top until all the moisture has been driven away 
and the lid thoroughly heated. The heating is not continued long 
enough to scorch the fish inside, however. After waiting a few 
minutes for the cans to cool, the vacuum is determined by means of 
a vacuum-pressure gauge. A hole is then made in the end near the 
seam by means of a hot, pointed, iron rod having a diameter of about 
6 mm. (% inch). By means of a sterile pipette, about 1 cc. of the 
liquid and finely divided particles of fish are transferred to a Petri 
dish and standard agar added. In a few cases 1 cc. of the liquid is 
also placed in anaerobic media. If any living organisms are found 
they are carefully examined to determine their nature. The presence 
of non-spore-formers indicates either a leaky can or contamination of 
the culture, the rigorous cooking to which salmon is subjected pre- 
cluding the possibility of their survival. After the bacteriological 
sample has been taken the top of the can is cut off just below the 
seam and the liquid drained into a 15 cm. (6-inch) white enameled 
pan and poured into a graduated cylinder. The solid portion of the 
fish is then examined. 

Vacuum. This is determined by means of a compound vacuum- 
pressure gauge equipped with a piercing point and a rubber gasket. A 
can should have sufficient vacuum, 8 inches (20.5 cm.) or more, to 
keep the ends concave under all conditions of temperature and alti- 
tude. Cans with tight seams will usually have some vacuum due to 
the absorption of oxygen by the salmon. Cans without vacuum will 
usually be found on close examination to have loosely rolled seams, 
a leak in the seam, or occasionally a small hole through the tin plate. 

Cleaning. No pieces of the gills, fins or intestines should be found 
in the can. All clotted blood which could be removed without tearing 
the flesh should have been washed out. 

Filling. There should not be more than three pieces of fish in 
the can and the long axis of these should parallel the long axis of the 
can. The ends of these pieces should be clean-cut, not jagged. 



202 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

Cooking. This is judged as sufficient or insufficient according to 
the friability of the bone. If vertebrae are present these are pressed 
between the thumb and fingers to determine this point. In the absence 
of vertebrae the brittleness of the smaller bones is tested. Insufficient 
cooking as determined by this method does not necessarily mean that 
the cans are not sterile, nor does a soft, easily friable bone insure 
sterility. This test is much used in the salmon industry, however, and 
is a useful indication. 

Salt. No definite amount can be stated since some people prefer 
more than others. However, there should be a definite salty taste. 

Pugh marks. If fish are pughed before the blood in them has 
clotted, blopd clots are formed along the puncture and may be found 
in the canned fish. They detract from the appearance of the pack and 
are an evidence of careless handling of the fish. 

Net weight. The food and drug law of the United States, as 
amended, requires that every package of food be correctly marked with 
the net contents. Several cans are weighed and the average net 
weight calculated. 

Oil. After the liquid has been standing in the graduated cylinder 
for a few minutes the oil rises to the top and the amount may be 
measured. When the fish begin their spawning migration they are 
usually fat, but since they no longer feed, this stored fat is gradually 
used up and the fish become poorer in quality. In deciding whether 
the oil in a can is good, average or poor, one must take into account 
the species, the section of the fish included in the can and the length 
of time the fish has been packed, since each of these factors has an 
effect on the amount of free oil m the can. 

Amount of liquid. The total amount of liquid, including the oil, 
is measured and recorded. As the fat in a living fish decreases the 
amount of water increases; therefore, if a large amount of liquid is 
found in the can the fish is likely to be of inferior quality. The 
character of the liquid is recorded as normal, slightly turbid or turbid. 
The liquid in cans containing badly decomposed fish may be "milky" 
in appearance. 

Color. Each species of salmon has its own characteristic color 
of the flesh, ranging from a deep red in the sockeye to a pure white 
in the white king. To a certain extent this color is modified in cook- 
ing, the degree of change varying with the different species. Further- 
more, the amount of color within each species varies greatly with the 



1922 C lough; on Indol and Skatol in Salmon 203 

stage of the spawning migration at which they are caught, since the 
color gradually fades as one of the physiological changes of this 
period. During decomposition the natural color of the cooked fish also 
appears to fade gradually. The color of the cooked fish is ex- 
pressed as good, average and poor, for that species. 

"Fresh water marking" on the skin. The skin of a salmon which 
is on the spawning migration takes on various colors, some of them 
very bright. Jordan and Everman (1896) speak of these colors as 
the "nuptial dress," but in the trade they are known as "water marks," 
since they usually become noticeable after the fish have reached 
brackish or fresh water. Distinctly "water-marked" fish are nearly 
always inferior to those which do not have these colors. 

Odor. This is one of the most reliable indications of decomposi- 
tion and is usually the factor which decides whether a can of fish 
shall be condemned as unfit for foot. In smelling the salmon it is 
first broken up between the hands and then held very close to the 
nose. The following terms, good, stale, tainted and putrid are the 
terms used in describing the odor. Fish which are canned while very 
fresh possess a normal fish odor. Fish canned when very slightly 
stale do not have this normal odor nor do they have a definite odor 
of decomposition. Both of these are recorded as good. Fish canned 
when stale will have abnormal odors, with a slight odor of decompo- 
sition, which usually leaves the fish during exposure to the air for 
a few minutes. These are recorded as stale, and are regarded as of 
poor quality but not unfit for food. Fish canned when tainted will 
have an unmistakable odor of decomposition, which persists in the fish 
even after exposure to the air for a few minutes. These are recorded 
as tainted and are regarded as unfit for food. When the odor of 
decomposition is very pronounced and offensive, apparent as soon as 
the can is opened, the odor is recorded as putrid. There are some 
odors which are encountered in canned salmon which, while abnormal, 
do not appear to be due to decomposition. Such an odor is found in 
"water marked" chum salmon. 

Texture. Each species when packed fresh has its own degree of 
firmness. Fish which are stale or tainted before canning have a text- 
ure which is more or less soft, and the degree of softness corre- 
sponds roughly to the amount of decomposition in the fish, however 
this must be interpreted with care since this texture is also affected by 
the fatness of the fish. 

Reddening. This is an important indication of spoilage. The flesh 



204 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

of raw salmlon takes on a feverish appearance, which persists through 
the processing; but when the can is opened the unnatural color will 
be found to be unevenly distributed and will fade quickly. It can 
thus be distinguished from the true color of the fish. 

"Honeycombing." The canned product sometimes has small holes 
in the flakes which may extend entirely through them. It is probable 
that these holes are a result of the gaseous condition of the partially 
decomposed flesh before canning. When a piece of this "honey- 
combed" flesh is placed on the end of the tongue a distinct biting 
taste is experienced similar to the "bite" of strong cheese. 

Turbidity of the liquid. The liquid in cans of salmon is, of course, 
always somewhat turbid, when the fish is stale or tainted before can- 
ning the turbidity is increased. Rough handling and very low tem- 
perature in the cans under examination may also result in a turbid 
liquid. 

Chemical examination. Cans which have been classed as strongly 
stale by physical appearance and odor may be examined chemically 
for indol and skatol to determine whether they should be condemned 
as unfit for food. Cans which are good, slightly stale or tainted, as 
determined by odor, need not be examined chemically as their status 
is already well established by the preceding tests. 

After carefully examining 48 cans (more for large parcels) ac- 
cording to the scheme outlined above, aided by an accurate record on 
forms similar to those in Fig. 1, the examiner is in possession of suffi- 
cient data to enable him to report on the thoroughness of the work- 
manship in packing the parcel, the quality of the fish when caught, 
their condition when canned and the adequacy of the cooking process. 

3. Chemical composition of fish fi^fsh 

One of the earliest investigators of the constituents of fish flesh 
was Morin, who in 1822 published his work on the composition of 
the smelt. Payen in 1854 determined the fat, protein, ash and water 
in the herring and the salmon. Several other chemists, among them 
Koenig (1876), Buckland (1874) and Almen (1887) also made 
similar analyses. The results obtained by them have been reviewed 
in detail by W. O. Atwater (1888) and recalculated to a common 
basis. The results which he and they obtained on salmon are given 
in table 3. The species is presumably the Atlantic salmon, Salmo salar. 



1922 



Clough; on Indol and Skatol in Salmon 



205 





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206 Publ. Puget Sound Biol. Sta. Vol. 3, No. 67 

Table; 3. Composition of the salmon. 



Year 


Author 


Water 


Solids 


Albuminoids 


Fats 


Ash 


Protein 
Nx6.25 


1854 


Payen. 


75.70 


24.30 


18.17 


4.85 


1.28 




1874 


Buckland. 


77.06 


22.94 


10.11 


7.11 


2.07 




1887 


Almen. 


70.33 


29.67 


18.06 


10.12 


1.49 


19.39 


1888 


Atwater. 


63.61 


36.39 


21.60 


13.38 


1.41 


22.39 



A considerable variation in composition will be noticed in table 3, 
which is probably due in large part to the seasonal changes in com- 
position. Recent work by Clark and Almy (1918), Green (1919) and 
Dill (1921) has shown marked changes in several species of fish, 
especially at the spawning period. The principal variations are in the 
fat and water percentages which are inversely related to each other. 
The protein and ash percentages do not change to any great extent. 
A few examples are given in table 4. 

Table; 4. Seasonal variation in the composition of fish. 



Author 


Fish 


Date 


Water 


Fat 


Protein 


Ash 


Clark and 
Clark and 

Dill 
Dill 


Almy Butterfish 
Almy Butterfish 

Yellow Fin Tuna 
Yellow Fin Tuna 


May 19 
Oct. 12 

May 14 
Sept. 8 


74.34 
69.99 

72.83 
69.17 


5.96 
13.42 

1.00 
6.54 


18.06 
18.25 

25.37 
24.00 


1.49 
1.40 

1.47 
1.32 



Cobb (1921a) gives a number of analyses of Pacific Coast salmon 
made by Atwater (1888), Langworthy (1898), Knisely (1908), 
Loomis (1912) and Elliott and Clemens (1916). Most of these 
analyses represent canned salmon, but Loomis also analyzed a fresh 
sockeye salmon. 

Recently Clark and Shostrom (Date?) have analyzed several hun- 
dred cans of salmon representing the five species of Pacific salmon 
from every important salmon canning district from northern California 
to the Yukon River in Alaska. The cans analyzed were all prepared 
from the second cut of an individual fish. This was done because 
the composition varies in different parts of the fish, and cans from 
the same part constitute a better comparison than those from different 
parts. Since this work was done in the National Canners' Laboratory 
at the University of Washington, Seattle, and has not yet been pub- 
lished, a summary of the results is given in table 5. 



1922 



C lough; on Indol and Skatol in Salmon 



207 



Tabld 5. Composition of Pacific salmon. 





No. individ 




Ether* 




Total 


Salt- 


Calories 5 


Species 


ual fish Moisture 


extract 


Protein 


ash 


free 


per 




analyzed 






(N x 6.25) 


ash 


pound 






% 


% 


% 


% 


% 




Chinook 


204 


63.53 


13.50 


19.48 


2.85 


1.18 


931 


(King) 
















Sockeye 


130 


64.52 


10.84 


20.67 


2.97 


1.29 


841 


(Red) 
















Coho 


99 


66.26 


9.47 


20.40 


3.15 


1.22 


778 


Pink 


90 


69.24 


6.16 


20.56 


3.47 


1.32 


642 


Chum 


120 


68.95 


7.42 


20.83 


2.40 


1.24 


700 


Average, all 


643 


66.50 


9.48 


20.39 


2.97 


1.25 


778 



Fish flesh consists of protein, fat, water, mineral matter and a 
very small percent of carbohydrates. The composition of such fat 
fish as the Pacific salmon compares very well with the meat of mam- 
mals, but in general fish flesh is higher in water and lower in fat than 
other flesh and in consequence has a lower fuel value. However, eaten 
with carbohydrate foods, a balanced diet is obtained equal in fuel 
value and muscle building power to a similar diet containing other 
flesh. Recent unpublished work by Barton and McMillan (Date?) 
indicates the presence of a very small amount of carbohydrate mate- 
rial in salmon flesh. 

The elementary composition of fish flesh was determined by 
Koenig and Splittgerber (1909) and compared with meat. Osborne 
and Heyl (1908) compared the hydrolytic products of halibut muscle 
with those of chicken muscle. Okuda (1919) has determined the 
clevage products of both ordinary flesh and "chiai" flesh of the bonito, 
Katsuwonas pelamis (table 6). The "chiai" flesh is the blood-colored 
flesh (dark meat) which occurs to a certain extent in the lateral 
muscle of most fish. The two kinds of flesh were separated as com- 
pletely as possible and subjected to hydrolysis with mineral acid. 
Results are given in per cent of the ash and moisture-free muscle 
substance. 

Okuda, Okimoto and Yada (1919) have published simlar work 
on the whale and the cod; Okuda, Uematsu, Sakata and Fujikawa 
(1919), and Okuda (1919a), on the spiny lobster and the cuttlefish. 

A great deal of disagreement occurs in the literature as to the 
amount of the different bases and amino-acids which are present in 

4This represents fat. 

SCalculated by the factors of Rubner : 18.6 Calories for 1 percent protein, and 42.2 
Calories for 1 percent fat, on the basis of 1 pound (454g). 



208 



Publ. Puget Sound Biol. Sta. 



Vol. 3, No. 67 



Table 6. Products of hydrolysis of "chiai" and ordinary fish 

muscle. 



"Chiai" muscle Ordinary muscle 

Alanin 1 - 1 2.3 

Valine 1-8 2.8 

Leucine 9.2 10.4 

Proline 3.0 3.1 

Phenylalanin 1-6 4.1 

Aspartic acid 3.2 3.3 

Glutaminic acid 12.1 8.1 

Tyrosine 2.9 2.1 

Arginine 7.08 7.8 

Histidine 3.16 3.04 

Lysine 6.78 7.41 

Tryptophane Present Present 

Glycocoll Not found Not found 

Serine ? ? 

Ammonia 0.78 0.64 

Guanine 0.12 0.09 

Adenine 0.1 0.04 

Hypoxanthine 0.03 0.08 

Xanthine ? ? 

Creatine 0.29 0.44 

Methylquanidine 0.005 

Taurine 0.34 

Creatinine Present Present 

Inosinic acid .013 .043 

Lactic acid .067 .062 

fish flesh. The methods used are rather difficult to carry out, and 
inexact, and it may also be tha'.. the amount in the flesh varies from 
time to time. The following results for creatin and creatinin in the 
flesh of the salmon will illustrate this. 



Author Creatin Creatinin 6 

Koenig and Splittgerber (1909) 0.027 0.207 

Suzuki and Yoshimura (1909) 0.320 None 

Okuda (1912) 1.525 0.182 

Cholin, neurin and muscarin have not been isolated from fresh 
fish, but cholin was found by Bocklisch (1885) in herring brine, and 
by Morner (1897) in "surfisk," a pickled, fermented fish product. 
Betaine has been found in the cuttlefish (Suzuki and Yoshimura 
1909), the cod (Yoshimura and Kanai 1913) and the shark (Suwa 
1909). Carnosin was found in the dried muscle of several fish, among 

GGrams per 100 grams of dried fish. 



1922 Clough; on Indol and Skatol in Salmon 209 

them salmon (0.055%), by Suzuki and Yoshimura (1909). Urea has 
been found in large amounts in the muscle of several fish, notably 
the dogfish and skate (Benson 1920), while work in our laboratory 
indicates that the flesh of the barracuda, and the atka fish (from the 
Aleutian Islands) also contains urea. Taurin (Suzuki and Yoshimura 
1909) and lactic acid (Liebig 1874) have also been found in small 
amounts, while according to Schondorff and Wachholder (1914) the 
glycogen content of fish muscle varies from to 0.59%. The carp 
at death was found to have 0.527%, after one hour 0.359%, after 
one day 0.145%, and on the third day none. The muscle of fresh 
salmon and cod contained none. In fish livers the glycogen varied 
from 2.5 to 12.94%. 

Oils are obtained both from the flesh of fish (fish oil) and from 
the liver (liver oil), but those fish which contain large amounts of 
fat in the liver have a small percentage of fat in the flesh. Little is 
known as to the composition of these oils. Stearic and palmitic acids, 
according to Lewkowitsch (1914), have been isolated from cod liver 
oil, but no oleic acid was found. Highly unsaturated acids are present 
but these are not identical with linolenic. Heyerdahl (quoted by 
Lewkowitsch 1914) concluded that the mixed fatty acids of cod liver 
oil contained about 4 per cent of palmitic acid, 20 per cent of jecoleic 
and 20 per cent of therapic acid. Liver oils contain cholesterol and 
other unsaponifiable matter. Fish oils contain palmitin. The mixed 
fatty acids contain highly unsaturated constituents which are not 
identical with linolic or linolenic acids. Tsujimoto (quoted by Lew- 
kowitsch 1914) found clupanodonic acid to the extent of 6 to 9 per 
cent. Fahrion (1893) is of the opinion that he has proved the pres- 
ence of jecoric acid in fish oil. 

Beal and Brown (1921) have recently made a study of the fatty 
acids of five commercial fish oils, among them salmon oil. They 
found evidence of the presence of myristic, palmitic and clupanodonic 
acids and also for the presence of acids more highly unsaturated and 
of greater molecular weight than clupanodonic, such as hexadecatri- 
enoic, arachidonic, eicosapentenoic, docosapentenoic, and docosahex- 
enoic. 

4. Decomposition of fish fi^sh 

When any organic material containing albuminous substances, 
such as fish, is allowed to stand under suitable conditions of moisture 
and of temperature, it decomposes very rapidly through the agency 
of enzymes secreted by various bacteria. This special fermentation, 



210 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

known as putrefaction, differs from natural digestion in that the for- 
mer yields many products not found in natural digestion, such as 
gaseous products, mercaptans, volatile acids, aromatic acids, amines, 
phenol, indol, skatol, and finally ptomaines. It was formerly thought 
that these products were excreted from the bacterial cells in which 
they had been formed, but it is now generally accepted that enzymes 
are secreted by bacteria, and these enzymes split the organic com- 
pounds, forming these and other new chemical substances. 

Many different species of bacteria may be concerned in a spon- 
taneous putrefaction. Bienstock (quoted by Rettger 1903, 1906) 
claimed that true putrefaction could be brought about only by obligate 
anaerobes. Rettger (1906) defined putrefaction as a bacterial decom- 
position of albuminous matter accompanied by the formation of 
"Faulnissprodukte." In his earlier work, Rettger (1903) took excep- 
tion to Bienstock's conclusions, but in later work (1908), using 
extreme care, his results confirmed those of Bienstock. Effront 
(1917) states that, "The putrefactive bacteria are ordinarily anaerobic, 
like Bacillus putrificus coli; nevertheless there are also very active 
ones which are aerobic, like Bacillus coli communis." Moreover, the 
bacterial flora is not the same throughout the putrefaction, some spe- 
cies converting the original proteins into substances which other bac- 
teria can utilize, and frequently forming substances which are injurious 
to themselves. According to Effront, none of the putrefactive bacteria 
produce pepsin, but several produce trypsin which converts the albu- 
minoid material into albuminoses, peptones and amino-acids, which 
are then attacked by erepsin, secreted by other species, and changed 
into simpler compounds. Finally, amidases come into action and 
bring about the formation of volatile acids and amines, as well as 
phenol and indol derivatives. 

The following substances are given by Effront as likely to occur 
in the course of putrefaction in addition to gaseous products (C0 2 , 
CH 4 , N 2 , H 2 S, PH 3 ) and residual peptones: "(1) Ammonia and 
amines; ethylamine, propylamine, and trimethylamine. (2) Volatile 
acids, comprising all the members of the fatty series up to caproic 
acid. They are sometimes normal acids, sometimes their isomers ; 
propionic acid is less frequent than the others ; formic acid is quite 
rare; acetic and butyric acid are especially common. (3) Aromatic 
acids and oxyacids; like phenylpropionic, oxyphenylacetic, and oxy- 
phenylpropionic acids. (4) Phenol, indol, scatol, pyrrol and its de- 
rivatives, these bodies sometimes being in very small quantities, or 
even completely absent. (5) Sulphur derivatives, like methyl-mer- 
captan. (6) Various amino-acids; leucin, tyrosin, tryptophane, and 



1922 Clough; on Indol and Skatol in Salmon 211 

sometimes glycin, creatinin, etc. (7) Various ptomaines; like put- 
rescin and cadaverin, the guanidins, cholin and neurin, pyridin, hydro- 
collidin, etc." 

In addition to the products formed by the splitting of nitrogenous 
compounds, there will also be decomposition products of the fats, con- 
sisting of glycerin and various fatty acids. The following reactions 
show how some of the above substances may be formed during de- 
composition. 

The effect of amidases on amino-acids was studied by Effront 
(1911), who gives the following reactions: (1). The monobasic acids 
are transformed into ammonium salts, e.g., glycin with the addition 
of hydrogen gives ammonium acetate. (2). Betain by the addition of 
hydrogen and the loss of water is transformed into the acetate of 
trimethylamine. (3). The polybasic acids undergo a molecular de- 
gradation, e.g., aspartic acid by the addition of hydrogen yields am- 
monium propionate and carbon dioxide. 

Tanner (1917) gives three methods by which straight chain acids 
may be simplified : ( 1 ) . Deaminization ; glycin to acetic acid and 
ammonia. (2). Decarboxylation; glycin to methylamin and carbon 
dioxide. (3). Oxidation; acetic acid to carbon dioxide and water. 
Tanner also shows by reactions the probable steps in the formation of 
phenol from tyrosin : (a) Tyrosin ; (b) parahydroxyphenylpropionic 
acid; (c) hydroxyphenylacetic acid; (d) paracresol; (e) phenol. 

Ehrlich (1909) has shown that amino acids may be fermented into 
alcohols corresponding to the acids used, ammonium bicarbonate being 
formed simultaneously. The acids which are thus formed may be 
very quickly changed to simpler ones ; glutamic acid, which probably 
first forms oxybutyric acid, yields succinic acid as a final product. 

Hopkins and Cole (1903) give the following steps in the decom- 
position of tryptophan by bacteria: (1) Tryptophan; (2) Indolpro- 
pionicacid; (3) Indolacetic acid; (4) Skatol; (5) Indol. The decom- 
position outlined above is the probable source of the indol found in 
our raw and canned salmon experiments. 

Effront (1917) says that tyrosin by the addition of hydrogen may 
yield methane and ammonium-p-oxyphenylacetate, a reaction which 
may explain the formation of methane from albuminoid material. 

Ornithin may give rise to either amino-valeric acid or to tetra- 
methylenediamine ; in the first case by the addition of hydrogen and in 
the second by the loss of carbon dioxide. Eysin may be transformed 
to pentamethylenediamine and carbon dioxide. Another ptomaine, 



212 Publ. Puget Sound Biol. Sta. Vol.. 3, No. 67 

neurin, is derived by loss of water from cholin, which is the constit- 
uent base of the lecithins. 

According to Mathews (quoted by Tanner, 1919) cystine goes to 
cysteine, then to thioethylamin and finally to ethyl mercaptan. Tanner 
(1917) has shown that bacteria can produce hydrogen sulphide from 
cystine. 

Effront (1917) speaking of the gaseous products of putrefaction 
states that the digestive enzymes (pepsin, trypsin, and erepsin) do not 
have an appreciable effect on the sulphur contained in albuminoid 
material, but that putrefaction enzymes split it off in the form of H,S 
and the mercaptans. Methane can arise from the reduction of tyrosin 
and carbonic acid as a by-product in many of the reactions already 
given. Ammonia is also a by-product of many reactions. However, 
as to the mechanism by which nitrogen, hydrogen and hydrogen phos- 
phide are formed, nothing is known. He is inclined to attribute the 
formation of nitrogen to the reduction of nitrates rather than to 
albuminoid material. 

Effront points out that substances formed early in putrefaction 
may not be present at later stages. Some of the poisonous substances 
elaborated during decomposition may disappear later, and it is more 
dangerous to eat meat just beginning to putrefy than that which is 
completely decomposed. Duclaux states that phenol and indol may be 
formed and then decomposed during the course of putrefaction ; but 
Effront states flatk/ that "when we do not find indol present, it is 
because it was never formed." 

The reactions and data given above show that it is impossible to 
give a simple and unchangeable scheme for decompositions since "the 
quality and quantity of the products formed are dependent upon the 
nature of the acting enzymes, which, themselves, are functions of the 
species of bacteria present, of the albuminoid substance to be trans- 
formed, and also of the physical and chemical conditions of the 
medium." 

Fish flesh, as has been shown, contains most of the amino-acids 
and other nitrogenous substances just mentioned, and presumably all 
of the decomposition products listed may be formed from them under 
suitable conditions. The question arises as to which one of these pro- 
ducts can be used to the best advantage as an index of the amount of 
decomposition in a product such as canned salmon. Ammonia has 
been frequently used as a measure of decomposition and seems to be 
fairly satisfactory when dealing with raw materials; but when used 
with canned products, such as meat and fish, the results show a de- 
cidedly disturbing factor, i.e., the ammonia produced by the cooking 



1922 Clough; on Indol and Skatol in Salmon 213 

process during canning. On account of this factor the investigator of 
canned meats and fish is left in doubt as to the percentage of the 
ammonia present in canned goods which is due to the canning process, 
and the percentage which is due to decomposition before canning. 
Loomis (1912) determined the "ammoniacal nitrogen" in fresh and 
canned salmon by two methods and makes the following observation. 
"As all samples of canned salmon were in good condition and gave no 
indication of deterioration as far as the senses could detect it, the 
results on 'ammoniacal nitrogen' are also of interest, being two or 
more times greater in the case of the canned product than in the 
fresh fish." 

Weber (1921) made an experimental pack of sardines in differ- 
ent stages of spoilage and determined the volatile nitrogen as am- 
monia and amines both before and after canning. He concludes that, 
"The cooking received during sterilizing very greatly increased the 
amount of ammoniacal material in the packed fish." He further 
states that in the case of fish which had undergone an excessive de- 
composition his results indicate htat the determination of volatile 
alkaline material may be used to detect this degree of spoilage but 
that with lesser amounts of decomposition the method is of doubt- 
ful value. 

Bidault and Couturier (1920) state that the quantity of the 
ammoniacal compounds in canned meat is a function of the tempera- 
ture of sterilization. 

The amount of free fatty acid undoubtedly increases during 
decomposition. Weber made a large number of determinations in 
his work on the sardine, but from the fact that only a few of the 
results are given in his report, and that these are very inconclusive, 
we may conclude that little reliance may be placed on the method. 
No reference was found in the literature to the effect of the canning 
process on the free fatty acids of canned foods; however, since one 
of the principal methods of hydrolysis of fats consists in heating the 
fats under pressure in the presence of steam, it seems highly probable 
that some of the fat in salmon may be hydrolyzed during the canning 
process, since all the factors of steam, heat and pressure are present. 

Rohmann (1908) states that tryptophan, when heated, gives 
rise to indol and skatol. Salmon flesh contains tryptophan, and the 
question arises as to whether the canning process is severe enough to 
break it down to indol or skatol. Experiments herein recorded prove 
that no considerable amount of these compounds is formed during 
the ordinary canning process. In fact, the canning process, as shown 



214 Publ. Pugct Sound Biol. Sta. Voe. 3, No. 67 

by further experiments, does net appear either to increase or decrease 
the amount of these compounds already present in partially spoiled 
salmon. No references were found in the literature as to whether 
indol or skatol had been found in canned salmon, but work done by 
Houghton and Hunter (1920) of the U. S. Bureau of Chemistry, pub- 
lished several months after this investigation was initiated, showed 
that it was frequently present and might possibly furnish a means 
of detecting spoilage. 

From a consideration of the above information relative to am- 
monia, fatty acids, and indol and skatol, it appeared that the latter 
were the most promising decomposition products to use as an index 
of spoilage. It was therefore decided to investigate the presence of 
indol and skatol in raw and canned salmon, and to attempt to de- 
velop a qauntitative method of determination which should be avail- 
able as a check on the organoleptic examination of the latter. Some 
work was also carried out, using ammonia and fatty acids as indexes 
of decomposition. 

5. Development oe a method eor detecting decomposition by 
means oe indol, and skatoe 

A. Selection of suitable color tests 

From a study of the literature relating to the decomposition of 
nitrogenous matter it seemed that the formation of indol and skatol 
would form the most accurate index of the presence and progress of 
decomposition. The next step therefore was to proceed to select a 
method for the determination of these substances. Owing to the 
small amount present even in very advanced stages of decomposi- 
tion, this method must necessarily be based on a color reaction. 
Fortunately both indol and skatol give very marked colors in ex- 
tremely minute quantities. Numerous color reactions are recorded in 
the literature, and among them are the following. 

Indol 

a. Formaldehyde reaction (Konto 1906). To 1 cc distillate in 
a test tube add 3 drops of a 4% formaldehyde solution and 1 cc. of 
concentrated sulphuric acid. Agitate the mixture and observe the 
appearance of a violet-red color if indol is present. Konto states 
that indol may be detected in a dilution of 1 :600,000. Skatol gives 
a yellow or brown color. 



1922 Clough; on Indol and Skatol in Salmon 215 

b. Cholera-red reaction (Salkowski, 1883; Tobey 1906a; Hawk 
1918). To 5 cc of the distillate in a test tube add one-tenth its 
volume of a 0.02 per cent solution of potassium, nitrite and mix 
thoroughly. Carefully run concentrated sulphuric acid down the side 
of the tube so that it forms a layer at the bottom. Note the purple 
color. Neutralize with potassium hydroxide and observe the pro- 
duction of a bluish-green color. 

c. Nitroprusside reaction (Deniges 1908; Hawk 1918). To a 
small amount of the material under examination in a test tube add 
a few drops of a freshly prepared solution of sodium nitroprusside, 
Na 2 Fe(CN) 5 NO-j- 2 H 2 0. Render alkaline with potassium hydroxide 
and note the production of a violet color. If the solution is now 
acidified with glacial acetic acid the violet is transformed into a 
blue. 

d. Nitroso-indol nitrate test (Hawk 1918). Acidify some of 
the material under examination with nitric acid, add a few drops of 
a potassium nitrite solution and note the production of a red precipi- 
tate of nitroso-indol nitrate. If the material contains but little indol 
simply a red coloration will result. 

e. Vanillin-sulphuric acid test (Steensma 1906; Deniges 1908; 
Blumenthal 1909; Nelson 1916; Zoller 1920; Weehuizen, (date?). To 
5 cc of the solution add 5 drops of 5% solution of vanillin in 95% 
alcohol, 2.5 cc of concentrated sulphuric acid and mix. If indol is 
present an orange color will be formed. Test is sensitive to 1 part 
in 2 million. If skatol is present a violet color will be formed. Test 
is sensitive to 1 pail in 4 million. 

f. Para-dhnethylaminobenzaldehyde (Herter 1905; Steensma 
1906; Deniges 1908a; Von Moraczewski 1908; Blumenthal 1909; 
Baudisch 1915; Nelson 1916; Ingvaldsen and Bauman 1920; Zoller 
1920). To 5 cc of solution, add 2 cc of a 2% alcoholic solution of 
paradimethylaminobenzaldehyde, 10 drops of concentrated hydro- 
chloric acid and mix. After a few minutes, add 1 cc of chloroform, 
shake and allow chloroform layer to separate. If indol is present a 
purplish red color is formed. Test is sensitive to 1 part in 1,000,000. 
Skatol produces a faint bluish color in dilutions of 1 part in 100,000. 

g. Beta-naphthaquinone reaction (Herter 1905 ; Herter and Fos- 
ter 1905, 1906; Bergheim 1917; Hawk 1918; Zoller 1920). To a 
dilute aqueous solution of indol (1:500,000) add 1 drop of a 2 per 
cent solution of B-naphthaquinone-sodium-mono-sulphonate. No re- 



216 Publ. Puget Sound Biol. Sta. Vol.. 3, No. 67 

action occurs. Add a drop of a 10 per cent solution of potassium 
hydroxide and note the gradual development of a blue or blue-green 
color which fades to green if an excess of the alkali is added. Render 
the green or blue-green solution acid and note the appearance of a 
pink color. Heat facilitates the development of the color reaction. 
One part of indol in 1,000,000 parts of water may be detected by 
means of this test if carefully performed. 

h. Pine wood test (Hawk 1918). Moisten a pine splinter with 
concentrated hydrochloric acid and insert it into the material under 
examination. The wood assumes a cherry-red color. 

i. Oxalic acid (Morelli 1908). Oxalic acid either solid or in 
concentrated solution takes on a red color with indol or indol vapor. 
Blotting paper soaked with oxalic acid solution introduced into the 
incubator or hung over a culture dry or moist, reacts very sensitively 
for indol produced by the bacteria. 

j. Furfural (Escallon and Sicre 1906). Extract culture with 
chloroform; drive off chloroform from the extract; take up residue 
in a few drops of alcohol, warm with 3 cc of the furfural reagent 
(glucose 1 gram and HC1 5 cc, warm to boiling, make up to 100 cc 
with water). Indol gives a reddish orange color. 

k. Glyoxylic acid (Dakin 1906). To 1 cc of solution to be 
tested add 1 cc of a solution of calcium glyoxylate (containing 0.1 
mg per cc) and 2 to 2.5 cc pure sulphuric acid. Note color at 
zone of contact and then slowly mix. Red color. Indol may be 
detected in a dilution of 1:200,000 and skatol 1:1,000,000. 

1. Pyruvic aldehyde (Nelson 1916). To 5 cc of the solution to 
be tested add a small crystal of ferric sulphate and a few crystals of 
pyruvic aldehyde. A layer of concentrated sulphuric acid is then 
added and if indol is present a violet ring is formed. Indol may be 
detected in a dilution of 1:500,000. 

Skatol 

a. Dimethylaniline test (Nelson 1916). To 5 cc of the solution 
to be tested add a few drops of dimethylaniline and shake vigorously. 
Add about 4 cc concentrated sulphuric acid to form a layer at the 
bottom. Violet ring is formed in dilutions of 1 :1,000,000 or more. 
Color soluble in chloroform. Indol does not interfere. 

b. Para-dimethylaminobemaldehyde reaction (Hawk 1918). To 



1922 Clough; on Indol and Skatol in Salmon 217 

5 cc of the distillate or aqueous solution under examination add 1 cc 
of an acid solution of para-dimethylaminobenzaldehyde (made by 
dissolving 5 grams of para-dimethylaminobenzaldehyde in 100 cc of 
10 per cent sulphuric acid) and heat the mixture to boiling. A 
purplish-blue coloration is produced which may be intensified through 
the addition of a few drops of concentrated hydrochloric acid. If 
the solution be cooled under running water it loses its purplish tinge 
of color and becomes a definite blue. 

c. Glyceric aldehyde (Nelson "1916). To the solution to be 
tested add a drop or two of glyceric aldehyde and sulphuric acid. 
Skatol produces an intense red color, soluble in chloroform, while 
indol gives a yellow color insoluble in chloroform. 

d. Methy alcohol (Sasaki, date ?). To the solution to be 
tested add 3 or 4 drops of methyl alcohol and an equal volume of 
sulphuric acid. The acid must contain a trace of a ferric salt and 
the alcohol must be free from acetone. Reddish violet color produced 
with skatol. Indol does not interfere. 

e. Para-dimethylaminobenzaldehyde (Blumenthal 1909; Steens- 
ma 1906). Test as for indol. Reaction not as delicate. 

All of the methods given above except i, j, k and / for indol, 
and c for skatol, were tried experimentally ; from them Ehrlich's 
test for indol (/) and either Herter's test (b) or the dimethylaniline 
test (a) for skatol, were selected as the most suitable for our use. 
Tests for indol, except e, f and g, and tests for skatol, except a, b 
and e, are not sensitive enough to be used with amounts of lmmg 
indol or skatol. 

The vanillin test for indol (e) is extremely delicate but fre- 
quently gives abnormal colors. Many substances other than indol 
give a similar color with vanillin. Experiments showed that HC1 
might be substituted for H 2 S0 4 ; the test was fully as sensitive and 
the charring effect of the concentrated H 2 S0 4 on organic substances 
was avoided. This test was frequently used as a confirmation test. 
A number of substituted vanillins were tried as color reagents and 
compared with vanillin. They proved to be less sensitive than 
vanillin and were discarded. 

B. Modification of the selected color tests 

Bhrlich test for indol (/) being chosen as the one most suitable 
for our work on raw and canned salmon, experiments were under- 



218 Publ. Puget Sound Biol. Sta. Vol,. 3, No. 67 

taken to increase its delicacy. The amount of indol in stale salmon 
is very small and the test to be used for its detection and estimation 
must be extremely delicate. The experiments were along the follow- 
ing lines: 

The color of the glass of the test-tubes needs to be considered. 
It was frequently found that amounts of indol which should have 
given a definite color apparently gave little or none. On investigation 
it was found that some of the test tubes in use were made of glass 
which had a decided bluish-green color which served to mask partly 
or completely the faint pink color produced by small amounts of 
indol by either the Ehrlich or Vanillin test. Since we were attempt- 
ing to record amounts of indol as small as 0.2-0.3 mmg the test tube 
was a big factor. Only thin test tubes of nearly colorless glass and 
as nearly as possible of uniform size were used in the experiments 
on raw and canned salmon. 

P-dimethylaminobenzaldehyde dissolved in alcohol has a strong 
yellow color which tends to mask the pink color in faint indol tests. 
By reducing the amount of reagent this interference is partially elim- 
inated. Half a cc of the Ehrlich reagent was the amount selected 
as the best for small amounts of indol (2.0 mmg or less). 

Concentrated HC1 partially destroys the yellow color of the 
p-dimethylaminobenzaldehyde reagent which interferes with the pink 
indol color. Since the 10 drops usually used have only a small effect, 
larger amounts were tried. A part of the yellow color was destroyed 
and the pink color became much more prominent. The indol color 
must be estimated at once however since it also is destroyed by the 
acid on standing. One cc was the amount selected as best for use 
with small amounts of indol. 

The influence of heat on the color was observed. After several 
experiments with the indol test, it was found that the color developed 
much faster when the test tubes were heated than when left at room 
temperature. However the time of heating had to be short or the 
color was partially destroyed by the acid. Heating for 20 seconds 
appeared to be the most favorable treatment. 

A modified Ehrlich method is given here. Reagents: (a) Para- 
dimethylaminobenzaldehyde, 2 grams in 100 cc of 95% alcohol; (b) 
HC1, 600 cc concentrated plus 200 cc of water; (c) Chloroform, 
U.S. P. Method: To 5 cc of the water test solution add 0.5 cc 
reagent a and 1 cc reagent b. Place in boiling water bath for 
about 20 seconds, shaking vigorously, then place in ice water about 
one-half minute and extract with 1 cc reagent c. Comparison is 



1922 Clough; on Indol and Skatol in Salmon 219 

made with standards prepared in exactly the same way. Delicacy: 
With pure water solutions of indol, 0.2 mmg may be easily detected 
in 5 cc, a dilution of 1 :25,000,000. 

Herter's test for skatol (b) is a very delicate one for both skatol 
and indol, but as in the Ehrlich test, the yellow color of the 
p-dimethylaminobenzaldehyde tends to obstruct the faint pink or blue 
produced by minute quantities of indol or skatol. Experiments indi- 
cated that 0.5 cc of the reagent to 5 cc of the solution to be tested 
gave better results than 1 cc. With this amount of reagent a distinct 
pink or blue could be obtained with 0.5 mmg indol or skatol in 5 cc 
of test solution, a dilution of 1 : 10,000,000. The distinction between 
indol and skatol is very marked even at this concentration, whereas 
the colors produced by the vanillin test on dilute solutions are very 
much alike. Substitution of 10 per cent HC1 for 10 per cent H 2 S0 4 
in making up the reagent was found advantageous, since the sulphuric 
acid tended to char organic substances in the test solutions obtained 
from fish, obscuring the results of the test. 

A modified Herter's method is given here. Reagents: (a) Para- 
dimethylaminobenzaldehyde, 5 grams in 100 cc of 10% HC1 ; (b) 
Concentrated HC1 ; (c) Chloroform, U.S. P. Method: To 5 cc of 
the water test solution add 0.5 cc of reagent a and heat nearly to 
boiling. Add a few drops of reagent b, cool, add 1 cc reagent 
c and shake vigorously. Comparison is made with standards pre- 
pared in exactly the same way. Delicacy: Either indol or skatol 
could be easily detected, when present to the extent of 0.5 mmg in 
5 cc water, a dilution of 1 :10,000,000. 

The dim ethyl aniline test for skatol (a), as given in some unpub- 
lished work by the U.S. Bureau of Chemistry, prescribes sulphuric 
acid. Experiments showed that hydrochloric acid might be used 
instead and the charring effect of the sulphuric acid avoided. In this 
test heat should be used to bring out the color. Skatol gives a pink 
color; indol does not interfere unless present in very large amount. 

A modified dimethylaniline method is given here. Reagents: 
(a) Dimethylaniline, C.P. and recently redistilled; (b) Concentrated 
HC1. Method: To 5 cc of the solution to be tested add 5 drops of 
reagent a and shake vigorously. Add 4 cc reagent b and heat 
in a water bath. Delicacy: With pure water solutions of skatol, 
1.0 mmg may be easily detected in 5 cc, a dilution of 1 :5,000,000. 

C. Distillation of indol from salmon 
Indol and skatol, as well as some of the other products of decom- 



220 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

position, are volatile with steam; and the question as to how com- 
pletely they may be separated from such a material as fish by steam 
distillation at once arises. Zoller (1920a) has determined the per- 
centage of recovery of indol from culture solutions of different pH 
concentrations, and concludes that a slightly alkaline solution (pH 9) 
gives the highest percentage. Bigelow and Cathcart (1921) give 
the pH value of canned salmon as 6.25, and the five species appar- 
ently do not differ appreciably. Furthermore, salmon in various 
stages of decomposition have been found to differ very little in hy- 
drogen ion concentration. In order to ascertain the effect of reaction 
on the amount of indol recovered we made a number of distillations 
of water solutions of indol, using our regular method, with the 
exception that varying amounts of alkali or acid were added to the 
flask. The same amount of indol (12mmg) was used in each distil- 
lation, and 500 cc was distilled. The recovery in none of these cases 
was very satisfactory, but nearly twice as much was recovered from 
the alkaline solutions as from the acid. 

Zoller also states that a higher percentage of recovery is attained 
when the volume of the liquid in the distilling flask is reduced as far 
as possible. We made a number of distillations, in some of which 
the liquid in the distilling flasks was allowed to increase, and in some 
the volume was much decreased. Twelve mmg indol were used and 
500 cc distilled in each distillation. Reducing the volume increased 
the percentage of recovery. 

The effect of adding NaOH to the fish in the distilling flasK 
was next tried, but so much difficulty was experienced from frothing 
by the alkaline mixture, that this treatment was not considered 
feasible. Furthermore, Bigelow's work showed that canned salmon 
was nearly neutral, and correction of reaction was not as necessary 
as with more acid substances. 

The percentage recovery of added indol from flasks containing 
fish formed the subject of the next experiments. Some fresh salmon 
was obtained from the market, skinned, boned and ground very fine, 
becoming thoroughly mixed in the process. A blank run on this 
salmon indicated that it was free from indol. Varying amounts of 
this fish with varying amounts of water and added indol were distilled 
and the percentage of recovery determined. The results showed that 
the recovery from fish was fully as good as that from water alone. 
They also showed that most of the indol which can be recovered from 
amounts of the magnitude used in these experiments is reovered in 
the first 500 cc of distillate. 



1922 Clough; on Indol and Skatol in Salmon 221 

To determine at what stage of the distillation most of the indol 
formed in decomposed salmon comes over, a series of experiments, 
using fish in various stages of decomposition, was carried out. These 
fish had been packed after being out of water from one to six days, 
and an idea of their condition can be obtained by consulting the data 
on chum salmon (6, F, below). Two hundred grams of fish, with 
200 cc of water, was placed in the flask and distilled at a uniform 
rate. The distillate was collected in 100 cc amounts and the indol 
in each determined as carefully as possible. The liquid in the flask 
(water test solution), after the evaporation of ether, frequently 
required dilution, in order that the color produced might not be too 
intense. 

The results of these experiments showed that when small 
amounts of indol are present a. very large percentage of all that it 
is possible to distill from the fish or other material is obtained in 
the first 500 cc. With larger amounts the percentage grows smaller 
until, when using fish canned when 6 days old, the amount recovered 
in successive 100 cc portions was not strikingly less in distillate 7 
than in distillate 1. The fact that the percentage obtained in the 
first 500 cc was rather small in the case of large amounts of indol 
was not particularly disturbing, since it was proposed to use the 
method as a measure of decomposition only in those cases in which 
the sense of smell was in doubt, in which cases only small amounts 
of indol, up to 6 mmg or less, were likely to be found. Therefore, 
in working with salmon it appears that it is satisfactory to distill one 
portion of 500 cc without the addition of alkali. An effort should be 
made to decrease the volume of liquid in the distilling flask to as 
small an amount as will still leave the contents in a fluid condition. 

D, Extraction of indol from the distillate 

The 500 cc distillate is transferred from the 600 cc beaker to a 
liter separatory funnel and extracted once with 100 cc of ether. Both 
ethyl ether and petroleum ether were used and the former finally 
selected as the most suitable, for several reasons. It was more easily 
obtained than petroleum ether of good grade. Frequently the latter 
could not be obtained from the local supply house and it was neces- 
sary to distill ordinary gasoline and use the low-boiling fraction. 
Frequently petroleum ether bought as low-boiling (below 60° C.) 
proved to contain 40% or more boiling above that temperature. 
Furthermore, some samples of petroleum ether contained impurities 
which interfered with color reactions, particularly with the vanillin 



222 Publ. Pitget Sound Biol. Sta. Vol. 3, No. 67 

sulphuric acid reaction. Nearly every can of U.S. P. ethyl ether 
proved to be free from interfering substances and of course boiled 
at a constant low temperature. It was therefore used exclusively in 
the raw and canned salmon experiments. 

When the distillate was violently shaken in the separatory fun- 
nel with the ethyl ether, a persistent emulsion usually resulted, from 
which only a part of the ether could be separated and also only a 
part of the indol. A quantity of concentrated HC1 (10 cc) was 
therefore added to the funnel before shaking, which prevented the 
formation of an emulsion and gave a sharp separation of the ether 
and water. Care should be taken to use c.p. HC1, since there appears 
to be some substance in commercial HC1 which interferes with the 
color test. Although only about 50 per cent of the ether used in the 
first extraction separates from the water, practically all of the indol 
which can be recovered is secured in the first extraction. 

During decomposition other products than indol and skatol are 
formed, and since some of these may distill with steam and interfere 
with the color reactions for indol and skatol, the ether extracts of 
the distillate are washed with dilute NaOH (2.5%). Repeated 
experiments showed that washing with alkali alone rendered the 
water test solution alkaline and also interfered with the color reac- 
tion; however, if the ether was rewashed with dilute acid (10 cc 
concentrated c.p. HC1 in 200 cc water) no interference with the 
color reaction was experienced. Repeated experiments showed that 
washed ether extracts gave clearer and slightly more intense color 
tests than exactly similar unwashed ether extracts. 

E. Evaporation of the ether extract 

After the ether extractions have been washed with alkali and 
acid they must be evaporated over a small amount of water (the 
test solution). This evaporation may be allowed to take place spon- 
taneously ; or be hastened by a current of air drawing away the ether 
vapor ; or by heating, either on a hot plate or a water bath, or by im- 
mersion of the flask in hot water. All of these methods were tried in 
order to find which gave the least loss of indol. In each method 
the same quantity of ether (130 cc) received different amounts of 
indol in 15 cc of water and after evaporation the water was divided 
into three equal portions and the indol determined in each by a dif- 
ferent method. The results indicated that some method of evapora- 
tion by heat was preferable to spontaneous evaporation, and among 



1922 Clough; on Indol and Skatol in Salmon 223 

the heating methods that of the steam bath seemed to be the best by 
a slight margin. 

Table 7. Loss of indol during the evaporation of the ether extract. 



Indol 
Method added 



Indol recovered in mmg. 



Ehrlich's Herter's Vanillin Total 
mmg. test test test 



Spontaneous evaporation 


12. 


2.2 


1.8 


3.0 


7.0 


Immersion in hot water 


12. 


3.0 


1.8 


2.8 


7.6 


Hot plate 


12. 


3.0 


3.0 


3.8 


9.8 


Water bath and aspirator 


12. 


3.8 


2.8 


4.0 


10.6 



The temperature of the water test solution under the ether 
remains at about 40° C. until nearly all of the ether is gone, when it 
rapidly rises, and great care must be exercised at this point to take 
the flasks off the water bath while there is still a little ether left. 
The last traces of ether may be removed by rotating the warm flask 
and drawing air through it by an aspirator. All the ether must be 
removed before the test solution is divided for the color tests. 

The amount of water used for the test solution varies but is 
usually 10 cc. This amount enables one to make check color tests 
on two 5 cc portions ; or if the first 5 cc portion gives a color too 
intense for comparison with the standard colors, the second 5 cc may 
be diluted to any desired extent in order to reduce the amount of 
indol in 5 cc to about 3 or 4 mmg, which amounts give colors of 
maximum ease of comparison. Experiments were made which proved 
that such dilutions could be safely and accurately made. 

F. Color tests on the water test solution 

After the ether has been entirely removed from the flasks 
containing the test solutions, these are ready to be subdivided and 
the color tests made. 

Usually the water test solutions consisted of 10 cc which was 
equally divided in two thin-walled, colorless, glass test tubes, one 
used for the test and one held in reserve. The tubes were placed 
in a rack in numerical order; and a series of standard tubes con- 
taining 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 6.0 mmg indol in 5 cc 
of water arranged in another rack. A series of reagent burettes 
arranged on a revolving stand contained the following reagents : 
Water, standard indol solution, p-dimethylaminobenzaldehyde solu- 
tion, hydrochloric acid, chloroform, and sometimes a modified 
Herter's reagent and a standard skatol solution. A boiling water 



224 Publ Puget Sound Biol. Sta. Vol. 3, No. 67 

bath, having a basket of coarse wire screen inside to keep the test 
tubes from falling, and two beakers of ice water, completed the 
arrangements. Each tube received the reagents, was heated 20 
seconds in the boiling water, and was plunged into ice water to cool, 
while a second tube received reagents and heating; after which the 
first tube was moved to the second beaker, and a third tube received 
reagents and heating. As soon as this tube was ready for the ice 
water, the first tube was cool enough to be extracted with 1 cc of 
chloroform, when it was placed in its proper place in the rack. By 
following this routine, each tube, in both the standard and unknown 
solutions, received exactly the same treatment. As soon as all the 
tubes had been treated they were compared with the standard tubes 
by holding in front of a piece of white paper and the color in the 
chloroform estimated as accurately as possible. The results were 
recorded in terms of mmg, on a basis of 100 grams of fish used. 

Standard solutions of indol and skatol contained 4 mg per 
liter, and they were made up fresh about every day, for experiments 
proved that they lost strength very rapidly. Owing to the difficulty 
in weighing accurately so small an amount of indol as 4 mg, alcoholic 
solutions containing 80 mg in 100 cc were prepared. By pipetting 
5 cc of the strong solution intc a liter volumetric flask and filling 
to mark, a standard solution was easily and accurately prepared. Such 
concentrated solutions in alcohol, if tightly stoppered, will keep for 
months. 

G. The method as finally developed 

The substance to be examined is thoroughly mixed, by grinding 
in a meat grinder, if necessary, as in the case of raw salmon, and a 
sample weighing 200 grams transferred to a liter, round bottomed, 
long necked flask, using about 200 cc of water. A current of live 
steam is then passed through the mixture in the flask until 500 cc 
of distillate are collected. A gallon oil can, having a long glass 
safety tube placed in the opening on top and a rubber tube attached 
to the spout, makes a very satisfactory steam generator. The steam 
is passed through the fish by means of a glass tube reaching to the 
bottom of the flask (flask inclined at an angle of about 45°) and so 
bent near the end as to give a rotary motion to the contents as the 
steam issues from it. The flask is kept boiling hot by being placed 
in a boiling, saturated salt solution. The steam from the flask is 
received in a vertical worm condenser, the end of which projects 
below the surface of a small amount of water in the receiver (600 cc 



1922 Clough; on Indol and Skatol in Salmon 225 

beaker). The distillate is transferred to a liter separatory funnel, 
acidified with 10 cc c.p. concentrated HC1 and extracted with 120 cc 
ethyl ether (U.S. P.) with repeated and vigorous shaking of the 
funnel. After the ether has separated, the ether layer is transferred 
to a 250 cc separatory funnel and washed first with 25 cc of NaOH 
solution (2.5%) and then with 25 cc dilute HC1 (10 cc c.p. concen- 
trated HC1 plus 200 cc water). The first washing is to remove 
compounds which might interfere in the color tests, and the second 
to neutralize any alkali left in the ether. The ether is then placed 
in a small flask with 10 cc of distilled water and evaporated on a 
steam bath, taking great care that while the last of the ether is 
being driven off the water layer is not heated appreciably above the 
boiling point of ether, since the indol may be easily lost by volatiliza- 
tion at this stage. A 5 cc portion of the 10 cc water residue is now 
tested for indol, and 5 cc for skatol, by the modified tests before 
described (5, B). 

6. Experimental work on the five species of salmon in different 
stages of decomposition 

A. General outline of the method employed 

Having now developed a satisfactory method for the determina- 
tion of indol and skatol in salmon, a comprehensive study of raw 
and of canned salmon was planned for the purpose of determining 
the significance of the presence of these decomposition products. 
This study was to cover the physical changes, the appearance and 
increase of indol or skatol, and a qualitative and quantitative inves- 
tigation of the bacterial flora during progressive decomposition, 
together with such correlations among the physical, chemical and 
bateriologial changes as could be discovered. 

These experiments will first be described in general, and any 
divergence from the general method will be given in the portion 
devoted to each species of salmon. In these portions the physical, 
chemical and bacteriological condition of each species of fish at the 
time of inspection are given in daily average tables showing the 
progressive nature of decomposition under each factor studied and 
in charts giving the results of the indol tests in both raw and canned 
salmon. 

Dr. Carl R. Fellers, the bacteriologist of the laboratory, took 
charge of the bacteriological work, while Mr. Oscar E. Shostrom, 
assistant chemist, and I, were responsible for the physical and 



226 Publ. Puget Sound Biol. Sta. Voh. 3, No. 67 

chemical problems. Doctor Fellers has very kindly permitted me to 
include a part of the bacteriological results, in order to show the 
correlations between bacterial flora and indol and skatol content. 
The salmon used in these experiments were obtained as they 
were taken from the traps, in order that there might be no question 
as to their exact age out of water. They were placed in boxes hold- 
ing about 10 or 12 salmon each, and stored on the dock over the 
water, under cannery conditions as nearly as possible. In every case 
the traps were lifted early in the forenoon. The next morning and 
each succeeding morning (even periods of 24 hours each) one-sixth 
of the fish were brought from the dock to the laboratory by auto- 
mobile. A maximum-minimum thermometer was placed with the 
fish stored on the dock, and each morning the range of temperature 
for the preceding 24 hours recorded. The fish taken each morning 
were selected without regard to their condition, some from the top 
and some from the bottom of the boxes. Immediately upon arrival 
at the laboratory, each fish received a designating letter, was hung 
upon a board which had been painted white and divided into six- 
inch (153-millimeter) squares with black lines, and was photographed 
with an ordinary kodak having a portrait lens attached. The raw 
salmon were then examined as follows 7 : 

Physical examination. The salmon were laid in order on a large 
table, measured and weighed. Three of each species were again 
weighed after cleaning to show the percentage loss. The general 
appearance of the fish was then recorded as indicated below. 

The skin, as the fish is taken from the water, is bright and free 
from slime, but it gradually grows dull and much slime appears. This 
slime forms an excellent medium for the growth of bacteria and helps 
to distribute them from fish to fish. 

The scales adhere firmly at first but gradually grow looser. 

The gills are bright red at first, gradually growing gray and 
finally greenish gray. Frequently the gills on one side of a fish 
were red while, those on the other side were gray. 

The eyes are bright, transparent and slightly bulging, when the 
fish is taken from the water. They gradually become bloodshot or 
gray, and less prominent, finally becoming sunken ; in extreme cases 
only the socket may remain. 

7In the original typewritten thesis, deposited in the library of the University of Wash- 
ington, a separate page is devoted to each of the 138 fish examined, giving all the physical, 
chemical and bacteriological data obtained, together with a photograph of the fish. Owing to 
the expense of printing, these data on individual fi^h have been omitted. However, the data 
have been averaged by days and are presented in tables, thus showing the decomposition changes 
from day to day. 



1922 Clough; on Indol and Skatol in Salmon 227 

The elasticity changes. Shortly after death the fish becomes 
rigid in rigor mortis and the flesh is firm. This rigidity is lost after 
a few hours but the flesh remains firm for some time. The flesh 
is elastic ; where pressed in it immediately springs out again as soon as 
the pressure is removed. This response becomes slower and slower 
until finally impressions made with the thumb or finger remain. 

Fly-blows and maggots are sought. Flies soon gather about a 
pile of fish ; unless the heaps are well protected they receive masses 
of eggs. In about 24 hours these produce maggots, resulting in a 
very filthy condition. 

Bacteriological examination. The scales were scraped from a 
small area near the back, close to the dorsal fin, and a hot iron was 
pressed against the skin until it was thoroughly seared over an area 
of about three square inches (58.5 sq. cm). With sterile scalpels and 
needles an incision was made to a depth of an inch (2.5 cm) or 
slightly less, and a piece of flesh weighing five or six grams placed 
in a weighed sterile bottle (250 cc) containing broken glass and 
100 cc water. The bottle was weighed again and the exact weight of 
flesh taken determined. This process was repeated on the belly near 
the ventral fin. A small piece of the gills was also cut off with 
sterile scissors and placed in a bottle. The fish was then slit open 
and a piece of the intestine taken from just behind the stomach. (In 
the case of the king and pink salmon a piece of caecum was also 
taken and placed in a fifth bottle). The four bottles respectively 
containing weighed amounts of back flesh, belly flesh, gills and 
intestines were then shaken vigorously until the sharp pieces of 
glass had cut the tissues into very small pieces and the bacteria were 
as uniformly distributed in the liquid as possible. Dilutions were 
made, using sterile water, in order that the Petri dishes might not 
be thickly sown, and 1 cc of the diluted liquid placed in a Petri dish. 
The neutral dextrose agar used was composed of 5 g peptone, 5 g 
dextrose, 3 g beef extract, 15 g washed agar and 1,000 cc water. It 
was neutral to phenol red. The plates were incubated at 30° C. for 
72 hours and the colonies counted. Other cultural characteristics of 
the organisms found in the back flesh were studied; among them, 
growth in anaerobic media (Van Ermengem 1912), and fermentative 
ability in dextrose and lactose media (Am. Pub. Health Assoc. 1920). 
Characteristic representative colonies were transferred from the agar 
plates to agar slants for preservation. Some of the anaerobic tubes 
were also preserved. The indol producing ability of many of these 
bacteria was later determined (7, A). 



228 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

Further physical examination. The condition of the viscera was 
observed. As a rule no very striking changes took place for several 
days. Occasionally the viscera was found to be gray or bloody; after 
several days the viscera usually became soft, frequently broken, and 
in extreme cases partially liquified. In a few fish the belly walls 
were broken through so the viscera protruded. The reddening in 
belly walls is an index of condition. The normal color gradually 
gave place to a feverish red, which began at the gills and spread 
backward along the belly walls. The intensity of this abnormal color 
was greatest near the gills. This is a very characteristic sign of 
decomposition. It was found in practically every case after the fish 
had been standing 72 hours or more. 

Gas gathers under the membranes. Usually on the fourth day, 
sometimes on the third, small bubbles were noticed under the mem- 
branes which line the belly cavity. By the fifth and sixth days these 
became very numerous and probably were scattered all through the 
flesh, producing the condition in canned fish spoken of as "honey- 
combing." 

Loose ribs are an indication of condition. As the fish grew older 
the belly membranes grew weaker and the flesh became loosened 
from the bones in such a way that the ribs, during butchering and 
cleaning, frequently broke loose, from the belly walls. 

The odor of the gills, viscera, walls of the belly cavity, and 
flesh in the back, were carefully recorded. The descriptive terms 
used were: good, lack of odor, stale, taint and putrid; both stale and 
taint were frequently divided into three degrees, for example, slightly 
stale, stale, strongly stale. The flesh of all the fish 24 hours old had 
a good normal fish odor. Frequently the flesh of fish 48 hours old 
did not possess this characteristic odor nor did it have any odor of 
decomposition. In such cases we reported this condition as "lack 
of odor." Stale fish had abnormal odors, with a slight suggestion 
of decomposition while tainted fish had an unmistakable odor of 
decomposition; when this was extreme, the fish were spoken of as 
putrid. 

Chemical examination. The amount of indol in the gills, viscera 
and flesh was determined. The gills, together with the bony struc- 
tures to which they are attached, were ground in a meat grinder and 
thoroughly mixed. Under the term "viscera" we placed everything 
within the belly cavity, including eggs or milt. This was ground and 
mixed. Whenever the ground gills or viscera amounted to 200 
grams, this amount was taken for distillation. When there was 



1922 Clough; on Indol and Skatol in Salmon 229 

law- 
less than 200 g, as large a sample as possible was taken and the 
weight recorded in order that the indol might be calculated to the 
uniform basis of 100 g. After the dressed fish was thoroughly 
washed, portions of the flesh were distilled for indol. The portions 
selected varied with the different species and are described in the 
part devoted to each species. These determinations were made 
according to the method given in 5, G of this paper. 

Canning. The remainder of the dressed and cleaned fish was 
cut transversely into sections of suitable length for half pound 
(227g) cans. In the case of large fish, some of these sections were 
big enough to fill two cans ; in such cases the section was 
cut into two nearly equal pieces, and the can receiving the piece 
without the backbone was marked with a star. The cans were placed 
in order; and as soon as the covers were clinched on, were marked 
in such a manner as to identify the species, the individual fish, and 
the particular section of that fish. For example, the cans obtained 
from the coho salmon "A" were marked as follows : 

CA1 CA2 CA3 CA4 CA5 CA6 CA7 CAS CA9 
CA2* CA3* CA4* CA6* CA7* 

In every case from 8 to 8^4 oz. (227-234 g) of fish were 
placed in each can with about */£oz. (3.5g) of salt. All of the cans, 
except those containing the king salmon, were exhausted before cook- 
ing. For this purpose the covers were clinched on rather loosely 
and the cans placed in a pressure cooker with the steam issuing 
freely from the petcock. They remained in the live steam for 12 
minutes and were then tightly closed with a hand seamer. All of the 
cans were cooked at 10 pounds (4.54 kg) pressure (240° F. or 115.5° 
C.) for 80 minutes, then removed and allowed to cool on the cement 
floor. A few days after all of the fish of one species were canned, 
certain cans from each fish were carefully examined according to 
the method outlined in 2. The cans selected and the reason for their 
selection are considered in the parts devoted to the several species. 

B. Experimental work on king salmon 

Eighteen king salmon were obtained as they were taken from a 
trap in the San Juan Islands at 10 a. m., July 30. They were im- 
mediately placed in two large fish boxes, covered, and brought on a 
fish boat to the dock of the San Juan Fish Company at Seattle, where 
the boxes were stored near the sea end of the dock. The maximum 
and minimum temperatures in the 7 readings in the fish boxes during 



230 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

the 6 days of storage were, in centigrade degrees, 21.1 and 15.6, 23.3 
and 15.3, 21.1 and 16.1, 19.4 and 15, 19.6 and 15, 19.4 and 15, 20.9 
and 14.4; (in Fahrenheit degrees 70 and 60, 74 and 59.5, 70 and 61, 67 
and 59, 67.5 and 59, 67 and 59, 69.5 and 58). 

Three fish were brought to the laboratory each morning. Indol 
determinations were made on the gills, viscera and flesh. With a 
view to obtaining a sample which should represent the whole fish, 
pieces of flesh, each weighing about 250 grams, were taken from the 
two ends of the dressed and cleaned fish. These were ground to- 
gether, mixed, and two portions of 200 g each distilled for indol. The 
results indicate that an unfortunate selection of flesh was made. The 
first cut of each fish is likely to contain more indol than any other 
cut, due to the rapid bacterial invasion from the gills. The last cut 
also contains more than the average for the fish as a whole. There- 
fore, since these cuts were selected for the determination of indol in 
the raw fish, the results were bound to be much higher than those 
obtained in the cooked salmon using cuts less heavily invaded by bac- 
teria. The indol was determined in three cans from each fish rep- 
resenting the third, sixth and next to the last cuts. The first and 
last cuts had been used raw, and the second cut (canned) was saved 
for possible use in a series of food analyses to be made during the 
winter. The three cans selected gave an idea of the condition of the 
fish at points near the ventral fins, near the end of the body cavity, 
and near the tail. (See table 8 and Fig. 2). 

C. Experimental work on pink salmon 

Thirty-six pink salmon taken from a trap in the San Juan Islands 
about 9 a. m., August 17, were placed in boxes, covered, and brought 
on a fish boat to the dock of the San Juan Fish Company at Seattle, 
where the boxes were stored near the sea end of the dock. The max- 
imum and minimum temperatures in the fish boxes during the six 
days of storage were, in degrees centigrade, 16.1 and 11.7, 16.7 and 
12.2, 18 and 13.3, 19.4 and 13.6, 18 and 14.4, 18.9 and 14.4; (in de- 
grees Fahrenheit, 61 and 53, 62 and 54, 64.5 and 56, 67 and 56.5, 64.5 
and 58, 66 and 58). (See also table 9 and Fig. 3). 

Six fish were brought to the laboratory each morning. Flesh for 
the indol test consisted of a portion from each end of the fish to- 
gether with a portion, equal to the two other portions combined, con- 
sisting of an entire section of the fish in the region of the dorsal fin. 
These three portions, weighing altogether about 250 grams, were 
ground together and mixed. Owing to the small size of the fish, only 



1922 



Clough; on Indol and Skatol in Salmon 



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Fig. 2. Indol in king salmon before and after canning. 



1922 



Clough; on Indol and Skatol in Salmon 



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Fig. 3. Indol in pink salmon before and after cannins 



1922 Clough; on Indol and Skatol in Salmon 235 

one indol determination was made on the flesh. The results again 
indicated that the portions of the flesh were not wisely selected. 

D. Experimental work on sockeye salmon 

Thirty sockeye salmon were obtained as they were taken from a 
trap in the San Juan Islands about 9 a.m., September 3. They were 
immediately placed in boxes on the deck of the fish boat, covered, and 
brought to Seattle. Upon arrival early in the morning of September 4, 
five were taken at once to the laboratory, and the rest stored on the 
dock. The maximum and minimum temperatures in the fish boxes 
during the 6 days of storage were, in centigrade degrees, 16.7 and 
12.2, 16.7 and 10, 15.6 and 13.3, 17 and 11.7, 16.7 and 11.1, 16.7 and 
11.7; (in Fahrenheit degrees, 62 and 54, 62 and 50, 60 and 56, 62.5 
and 53, 62 and 52, 62 and 53). (See table 10 and Fig. 4). 

Five sockeyes were brought to the laboratory each morning. 
Flesh for the indol determination was selected in such a way as to 
show whether the cooking of the canned fish increased or diminished 
the amount of indol present. The first transverse section of the fish 
(called the first cut) consisting of about one pound (454 g) was 
divided as equally as possible into two parts, in one of which the indol 
was determined at once, and in the other after canning. A section of 
similar weight, cut from the fish just in front of the dorsal fin (usually 
the third or fourth cut) was treated in an exactly similar manner. 
The results gave a much closer agreement between the raw and cooked 
salmon than was obtained with the king and the pink salmon. 

E. Experimental work on coho salmon 

Twenty four coho salmon were obtained as they were taken from 
a trap in the San Juan Islands about 9 a.m., September 3. They 
were immediately placed in boxes on the deck of the fish boat, cov- 
ered, and brought to Seattle. Upon arrival, early in the morning of 
September 4, four were taken at once to the laboratory, and the rest 
stored on the dock. The maximum and minimum temperatures in the 
fish boxes during the 6 days of storage were, in centigrade degrees, 
16.7 and 12.2, 16.7 and 10, 15.6 and 13.3, 17 and 11.7, 17 and 11.7, 16.7 
and 11.1, 16.7 and 11.7; (in Fahrenheit degrees, 62 and 54, 62 and 50, 
60 and 56, 62.5 and 53, 62 and 52, 62 and 53). 

Four coho salmon were brought to the laboratory each morning. 
Flesh for the indol determination was selected as in the case of the 
sockeye salmon. The results gave a much closer agreement between 
the raw and the cooked salmon than was obtained with the king and 
the pink salmon. (See table 11 and Fig. 5). 



236 



Publ. Puget Sound Biol. St a. 



Voi,. 3, No. 67 



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238 



Publ. Puget Sound Biol. Sta. 



Voi,. 3, No. 67 



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1922 



Clough; on Indot and Skatol in Salmon 



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Fig. 5. Indol in coho salmon before and after canning. 



240 Publ. Puget Sound Biol. Sta. Vol. 3, No. 67 

F. Experimental work on chum salmon 

Thirty chum salmon were obtained from a trap at Meadow Point, 
near Seattle, about 8 a.m., October 22. They were brought to Seattle 
on a scow and unloaded at the dock about 10 a.m. All of the fish 
were taken at once to the laboratory and stored in a box having 
several shelves so that there might not be too much pressure on any 
of the fish. For two days the box was kept in the laboratory near an 
open window and was then moved outside. The maximum and mini- 
mum temperatures in the box during the 6 days of storage were, in 
centigrade degrees, 20.6 and 15, 21.7 and 16.7, 22.2 and 11.1, 14.4 and 
11.1, 17.8 and 11.1, 13.3 and 10.6; (in Fahrenheit degrees, 69 and 59, 
71 and 62, 72 and 52, 58 and 52, 64 and 52, 56 and 51.) 

Five fish were used each day. Flesh for the indol determination 
was selected as in the case of the sockeye salmon. The results were 
somewhat higher in the fish out of water 48 hours than the results 
obtained from any of the other species, probably due to the higher 
storage temperature. (See table 12 and Fig. 6). 

Indol in raw salmon at different stages of decomposition was quan- 
titively determined in 138 fish. Two determinations were made on 
each fish, with the exception of the pink salmon, on which but one 
was made. In all, 229 determinations were made. Although the re- 
sults have already been given they are rearranged here to bring out 
the correlation between indol content and odor; and for comparison 
with the results obtained with the same salmon when canned (table 
14) as well as those obtained with commercial cans of salmon (table 
15). The odor on which table 13 was based was the odor of the 
belly cavity. 

As the odor of decomposition increased in the belly cavity, the 
amount of indol in the flesh also increased. The percentage of deter- 
minations having 1.5 mmg indol or more per 100 g of salmon increased 
in proportion to the odor of decomposition. The agreement between the 
results given here and those given in table 14 is very good. The terms 
used here to designate odor have been defined in 2. Although it is 
recognized that stale fish is of inferior quality, it is usually considered 
as not unfit for food. In this and other tables, therefore, those fish 
or cans of fish classed as stale have been designated as "passed by 
odor as fit for food," while tainted fish or cans of fish have been 
designated as "not passed by odor." 

Indol in experimental packs of canned salmon was quantitatively 
determined in 269 cans of the five species. The results have already 



1922 



Clough; on Indol and Skatol in Salmon 



241 



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Publ. Puget Sound Biol. Sta. Vol,. 3, No. 67 











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Fig. 6. Indol in chum salmon before and after canning. 



1922 



Clough; on Indol and Skatol in Salmon 



243 



Table 13. Correlation between indol and odor in raw salmon. 



Indol mmg 


Number of 


cuts classed by odor 


as 




per 100 g 




Slightly 




Strongly 


Tainted 




salmon 


Good 


stale 


Stale 


stale 


or putrid 


Total 


0.1—0.5 


58 


10 


3 








71 


0.6—1.0 


4 


10 


4 








18 


1.1—1.4 





1 


2 








3 


1.5— 





10 


21 


12 


94 


137 


Total 


62 


31 


30 


12 


94 


229 


1.5— 


0.0% 


32.2% 


70.% 


100.% 


100.% 


59.8% 



Reported as good. Reported as stale. 

0.1—0.5 68 3 

0.6—1.0 14 4 

1.1—1.4 1 2 

1.5— 10 33 

Total 93 42 

1.5— 10.7% 78.5% 

Passed by odor as 
fit for food. 

0.0—0.5 71 

0.6—1.0 18 

1.1—1.4 3 

1.5— 43 

Total 135 

1.5— 31.8% 



Not passed by 

odor. 







94 

94 

100.% 



been given in the previous pages of this section but are rearranged 
here in order to bring out the correlation between indol content and 
odor. Furthermore, comparison with the raw salmon used, and with 
commercial packs of salmon, can be more easily made. 

The results show an increasing percentage of cans containing 1.5 
mmg (or more) per 100 g of salmon, with an increase in the odor of 
decomposition. A few of the cans classed as good (3.9%) contained 
as much as 1.5 mmg while practically all of those classed as strongly 
stale or tainted contained this amount or more. The percentage of 
cans containing indol to the extent of 1.5 mmg or more is much 
higher than in the case of commercial cans of salmon (table 15). The 
agreement between the raw salmon (table 13) and the experimental 
cans of salmon prepared from them (table 14) is very good. Why 
the commercial cans contained less indol is not known. It may be 
that the bacterial flora was different. Pugsley (date?) carried on a 
decomposition experiment similar to the one described in this section, 
using chum salmon which had been in cold storage for three months, 



244 



Publ. Pugei Sound Biol. Sta. 



Voi,. 3, No. 67 



Table 14. Correlation between indol and odor in experimental 
cans of salmon. 



Indol mmg 
per 100 g 
salmon 
0.0—0.5 
0.6—1.0 
1.1—1.4 
1.5— 
Total 
1.5— 





Slightly 




Strongly 


Tainted 




Good 


stale 


Stale 


stale 


or putrid 


Total 


46 


4 


2 


1 





53 


24 


6 


8 





1 


39 


3 


2 


3 





1 


9 


3 


7 


22 


24 


112 


168 


76 


19 


35 


25 


114 


269 


3.9% 


36.8% 


62.8% 


96.% 


98.% 


62.4% 



Reported as good. Reported as stale. 

0.0—0.5 50 3 

0.6—1.0 30 8 

1.1—1.4 5 3 

1.5— 10 46 

Total 95 60 

1.5— 10.5% 65.9% 

Passed by odor as 
fit for food. 

0.0—0.5 53 

0.6—1.0 38 

1.1—1.4 8 

1.5— 56 

Total 155 

1.5— 36.1% 



Not passed by 

odor. 



1 

1 

112 

114 

98.% 



and some which had not been in cold storage at all. The results were 
entirely different. As stated in 7, A, apparently all of the indol form- 
ing bacteria had been killed during cold storage, and both the raw 
salmon and the canned salmon prepared from them contained almost 
no indol even after five or six days' storage at room temperature ; 
whereas the raw salmon which had not been in cold storage, and the 
canned salmon prepared from them, contained large amounts of indol 
after the third day. This example shows what a big variation in de- 
composition products may result from different bacterial floras. 

Indol in commercially canned salmon was quantitively determined 
in 544 cans during a period of ten months. These cans were drawn 
from 168 separate parcels of salmon, many of which were of rather 
poor quality. For this reason the proportion of stale and. tainted cans 
is very much higher than is found in the average parcel of 
canned salmon. In all 1,897 cans were opened for examination and 
544 tested for indol. In some of the samples the first 12 cans opened 



1922 



Clough; on Indol and Skatol in Salmon 



245 



were used for the indol test without regard to their condition, while 
in many samples only those which were classed as stale or tainted 
were used. This is an additional reason why the proportion of stale 
and tainted cans is so large in table 15. 



Table; 15. Correlation between indol and odor in commercial 
cans of salmon. 



Indol mmg 
per 100 g 
salmon 
0.0—0.5 
0.6—1.0 
1.1—1.4 
1.5— 
Total 
1.5— 



Good 
110 

77 
7 

11 
205 

5.36% 



-Number of cans classed by odor as 



Slightly 
stale 
45 
55 
7 
11 
118 
9.3% 



rongly 


Tainted 




stale 


or putrid 


Total 


5 


4 


212 


12 


15 


236 


3 


2 


27 


7 


22 


69 


27 


43 


544 



Stale 
48 
77 
8 
18 
151 
11.9% 25.9% 51.1% 



12.6% 



Reported as good. Reported as stale. 

0.0—0.5 155 53 

0.6—1.0 132 89 

1.1—1.4 14 11 

1.5— 22 25 

Total 323 178 

1.5— 6.81% 14.0% 

Passed by odor as 
fit for food. 

0.0—0.5 208 

0.6—1.0 221 

1.1—1.4 25 

1.5— 47 

Total 591 

1.5— 9.38% 



Not passed by 

odor. 

4 

15 

2 

22 

43 

51.1% 



The results show an increasing percentage of cans containing 1.5 
mmg (or more) of indol per 100 g of salmon, with an increase in the 
odor of decomposition. However, a few (5.36%) of the cans classed 
as good would be condemned on the basis of 1.5 mmg of indol, while 
nearly half (48.9%) of those condemned by odor would be passed by 
the indol test. As stated in 6, G, almost none of the cans (from our 
experimental packs) prepared from fish out of water 48 hours or less, 
gave a test for more than 1.5 mmg indol, and in those cases the raw 
fish showed signs of decomposition. It is evident, therefore, that 
although these few cans had an odor classed as good, considerable de- 
composition had already taken place. As pointed out in 7, A, many 
bacteria do not produce indol, although they may bring about a putre- 



246 Publ. Puget Sound Biol. Sta. Voh. 3, No. 67 

faction of the fish. This may explain why such a large percentage of 
tainted commercial canned salmon contained less than 1.5 ramg indol. 
The experimental pack of salmon as shown in table 14 contained more 
indol than the commercial cans examined. The difference may be 
due to a difference in bacterial flora between the waters of Puget 
Sound, where the fish for the experimental pack were caught; and 
the waters of Alaska, where most of the fish for these commercial 
cans were caught. There may be a larger percentage of indol formers 
in the flora of the waters of Puget Sound. 

In conclusion it may be said that when indol is found in amounts 
of 1.5 mmg or over per 100 g it is certain that a considerable amount 
of decomposition has taken place. However, we cannot safely accept 
the absence of indol as conclusive evidence of the absence of de- 
composition. In other words, a positive test has considerable value in 
judging the condition of canned salmon as regards decomposition, 
vvhile a negative test is of little value. 

G. Discussion of the results 

The results obtained in the experimental work on the five species 
of salmon have been summarized for each species, in tables and figures, 
in the previous pages of part 6. Furthermore, all of the condi- 
tions and changes studied during this investigation have already been 
discussed to some extent in the general outline of the experiment. 

The physical results on each of the species are very similar. All 
of the indications of decomposition increased in intensity from day to 
day, and although there were individual fish or cans which did not 
follow the general rule, the averages for the fish examined from day 
tc day showed a consistent and regular increase in each of these indi- 
cations ; this is shown in the tables giving the average daily decompo- 
sition changes. The chum salmon showed more rapid increase in de- 
composition changes than the others, and this was probably due to the 
higher temperature at which they were stored during the first two 
days. In the pink salmon, on the other hand, decomposition pro- 
ceeded much more slowly ; and this again was apparently due to the 
somewhat lower temperature prevailing at the beginning of the experi- 
ments on pink salmon. 

The chemical results from the different species are also similar, 
but here, too, the influence of the storage temperature is very marked, 
some of the chum salmon giving a test for 1.5 mmg or more of indol 
at the end of 48 hours, while none of the pinks gave a test for this 
amount until after 96 hours. In the case of all the species, however, 



1922 Clough; on Indol and Skatol in Salmon 247 

the amount of indol increased from day to day ; and although there 
were some determinations both of the raw and of the canned salmon 
which did not follow the general rule, yet the averages for the fish 
examined each day showed a rather consistent increase. The curves 
which are given for each species show that during the first two or 
three days little indol is found ; but after it begins to appear in notice- 
able quantities, the increase is very rapid, or, in other words, the curve 
bends sharply upward. The curves showing the results obtained from 
the gills do not show the pronounced initial lag shown by the curves 
for the flesh, either raw or canned, and this is no doubt due to the 
fact that the gills contain many bacteria at the beginning of the storage 
period, while the flesh is free from bacteria. Indol production, there- 
fore, may start at once in the gills, but not in the flesh until after it 
has been invaded by the bacteria. This invasion, as shown by the 
bacteriological results, is rather slow for the first 48 hours ; this agrees 
with the indol production. The curves for the viscera in most of the 
species show a longer initial lag than those for either the flesh or the 
gills ; and the amount of indol in the advanced stages of decomposi- 
tion of the viscera was less in nearly every case than in the flesh. It 
'may be that the viscera are a poor medium for the growth of bacteria 
and the formation of indol. Furthermore, the presence of the milt 
and roe (which are very slow in showing signs of decomposition) in 
the ground viscera served to dilute the more easily putrescible parts, 
such as the intestines, stomach, heart, etc., and thus lowering the per- 
centage of indol. Since the bacteriological samples were taken from 
the intestines only, while the chemical samples included the entire 
contents of the belly cavity, is it not surprising that the results do not 
agree very well. 

The curves given in Figs. 2 and 3 for king and pink salmon re- 
spectively, do not show a good agreement between the raw and the 
cooked flesh ; the raw flesh has more indol than the cooked. As pointed 
out elsewhere, this is due not to loss of indol in the canning process, 
but to an unfortunate choice of samples for this determination ; the 
raw salmon used was taken from those parts of the fish most heavily 
invaded by bacteria, while the canned salmon used represented parts 
much less heavily invaded. The curves given in Figs. 4, 5, and 6, for 
sockeye, coho and chum salmon, respectively show very good agree- 
ment between the raw and the cooked flesh. This is due to the fact 
that the raw and canned flesh used for the indol determination came 
from the same cut of the fish, one half being used raw and the other 
half canned. 

The variation in the amount of indol found in different parts of 



248 



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Fig. 7. Composite results for indol in sockeye, coho and chum salmon. 



1922 Clough; on Indol and Skatol in Salmon 249 

the same fish is shown in Fig. 7, which gives composite curves showing 
the results from the first cut and those from a cut near the dorsal 
fin. The first cut contains more indol because it is near the gills 
where it is quickly and heavily invaded by bacteria. These composite 
curves show excellent agreement between the results from the raw 
and the cooked flesh, espcially in the first cut. Why slightly more 
indol was found in the cooked than in the raw flesh is not clear, but 
it may be that the cooking process serves to break down the cell 
structure, liberating the indol, resulting in a more rapid and complete 
distillation. 

Figure 8 shows the results from the raw flesh of the five species. 
The difference in temperature prevailing at the time these different 
species were under examination may have been partly responsible for 
the difference in the results obtained. The maximum and minimum 
temperatures were taken each day, and these have already been given ; 
but for convenience, curves showing the mean temperatures from day 
to day are also given in Fig. 8. Inspection of these curves shows that 
the king salmon was stored at the highest average temperature and 
the coho and sockeye at the lowest. The king salmon contained the 
most indol, while the sockeye and coho had the least, with the excep- 
tion of the pink, which had less during the first four days. The chum 
salmon curve follows that of the king very closely at first ; but the 
rate of increase falls off, and the falling off is coincident with a sharp 
lowering of the temperature when the chum salmon were moved from 
the laboratory to the outside of the building. The low temperature 
prevailing at the beginning of the experiments with pink salmon is no 
doubt partially responsible for the small amount of indol found during 
the first four days ; while the increasingly higher temperatures toward 
the end of the storage period, together with their small size, may 
account for the rather rapid increase in indol content during the fifth 
and sixth days. Since the sockeye and coho salmon experiments 
were made at the same time, the temperature was not responsible for 
the difference in indol content. It is possible that this difference is 
due to the difference in the average size of the two species ; the coho 
salmon is much larger, so the bacteria would be somewhat longer in 
invading all parts of the flesh, and the proportion of indol in the flesh 
would be less. Temperature is without doubt a big factor in the 
spoilage of salmon, and the correlation between it and indol forma- 
tion is quite apparent from a study of the curves in Fig. 8. 

Skatol was not found in any of the raw salmon, nor in the cans 
prepared from them ; but indol was found in every test made on salmon 
which had been held for 48 hours or more. An organism capable of 



250 



Publ. Puget Sound Biol. Sta. 



Voh. 3, No. 67 





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Fig. 8. Correlation between storage temperature and amount of indol. 



1922 Clough; on Indol and Skatol in Salmon 251 

producing skatol was however isolated from the king salmon (7, A). 

The bacteriological results are very similar to the chemical re- 
sults, as might be expected, since the chemical substances formed 
during decomposition are due to the bacteria present. However, as 
is pointed out in a later section, not all bacteria produce indol, and 
for this reason the correlation between numbers of bacteria and indol 
content may or may not be a close one. The number of bacteria 
increased from day to day in each of the parts of the fish examined; 
gills, viscera, back flesh and belly flesh. The gills contained a large 
number of bacteria when the fish were drawn from the water, while 
it is probable that the other three parts of the fish examined were 
free from bacteria. At the end of 24 hours the gills usually contained 
several thousand bacteria per gram while the viscera sometimes and 
the flesh usually was sterile or contained very few bacteria. 

The correlation between the bacteriological results and the chem- 
ical results has been shown in the tables containing the average daily 
decomposition changes of the five species of salmon. For convenience 
these daily averages have been composited and the results expressed 
in curves in Fig. 9. In general the correlation is very good ; the curves 
for indol and for bacteria, in both the gills and the viscera, have very 
nearly the same shape throughout. It will be noted that the belly 
flesh is more rapidly invaded than the back, and this is probably due 
to the fact that the layer of flesh in the belly is much thinner than 
that in the back. The curve for indol in the raw flesh falls between 
the curves for bacteria in the belly and back flesh, due to the fact 
that the raw flesh was made up of both belly and back flesh. The 
sharp falling off in the number of living bacteria between the fifth and 
sixth day indicates that the media had become unfavorable, at least 
to some of the species of bacteria, with a consequent heavy mortality. 

From a consideration of the results obtained in this study of the 
decompositon of salmon, and from results which were later obtained 
in a study of the intestinal bacterial flora, it is believed that the spoil- 
age of fish taken while on the spawning migration proceeds primarily 
from the outside of the fish and not from the digestive tract. Bacteria 
invade the flesh from the gills, the anus, and the skin. In the case of 
fish caught while still feeding (by trolling), spoilage will proceed 
from both the outside and the inside. Correlations between the odor 
and the indol content in the raw salmon, in our experimentally packed 
salmon, and in commercial packs of salmon, have been given on pre- 
vious pages. Correlations between the bacteriological and the chemical 
results have also been shown, as well as between the indol content 
and the temperature of spoilage. Correlations between the tempera- 



252 



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Fig. 9. Correlation of bacteriological and chemical results. 



1922 Clough; on Indol and Skatol in Salmon 253 

ture, the indol content, the number of bacteria present, and various 
signs of decomposition, such as reddening, texture, etc., might be 
shown in a similar way; but enough has been written to show that 
the physical, chemical and bacteriological changes proceed simultane- 
ously and approximately parallel, and that the rate is dependent to a 
large extent upon . the temperature. Although the indol test cannot 
supplant the examination by odor and other physical signs of decom- 
position, it forms, nevertheless, a very useful check on the other 
methods of examination, and affords considerable information as to 
the history of the salmon under inspection. 

7. Formation of indol by various means 
A. Indol formation by bacteria 

So many cases were encountered in the study of both raw and 
canned salmon, in which the odor and the indol content were not in 
accord, that it seemed advisable to make a study of the bacteria which 
Dr. C. R. Fellers had isolated during the above investigation, to as- 
certain how large a percentage of them would produce indol in Dun- 
ham's peptone solution. In many cases cans classed by odor as 
"slightly stale", or even as "good", were found to contain as much 
indol as other cans which were classed as "tainted" ; and on the other 
hand, cans having a tainted odor sometimes did not give a test for 
either indol or skatol. Since the foul odor must be due to bacterial 
decomposition, it is evident that spoilage may proceed without the 
formation of these specific products. Rettger (1906, 1908), in his 
work on putrefaction, states that true putrefaction is due to strict 
anaerobes alone, and indol is seldom formed. Furthermore, Bacillus 
botulinus, a strict anaerobe, was inoculated for experiment into some 
sterile cans of salmon, then the cans exhausted and again sealed. Af- 
ter standing about two days at the temperature of the laboratory, the 
cans showed a pronounced swelling; on opening, they were found to 
possess a putrid odor; in fact, the contents of each can was in a 
liquid condition and bubbling with gas. No indol was found in any 
of these cans. 

The bacteria used in the determination of indol production 
were derived from the following five sources: 1. Isolated 
from the five species of salmon (raw) during the study of decompo- 
sition. 2. Isolated from commercially canned salmon. 3. Taken by 
the writer from raw salmon (gills, viscera, flesh and pugh marks) at 
various points in central and southeastern Alaska. 4. Taken by Dr. 



254 



Pitbl. Puget Sound Biol. Sta. 



Voi,. 3, No. 67 



C. R. Fellers from four species of salmon (raw) at Blaine. Washing- 
ton. Taken from points just in front of and just behind the stomach, 
to ascertain whether the intestinal tract of salmon on the spawning 
migration is sterile. A few cultures were also made from the gills 
and pugh marks. 5. Taken from chum salmon which had been held 
frozen in cold storage for three months. 

The method of experimentation was to inoculate the bacteria into 
10 cc of Dunham's peptone media and incubate at 37° C. for one 
week. The tube was then emptied into a 250 cc Fry flask and washed 
out with 40 cc of water. A current of steam was passed through and 
100 cc of distillate collected. This was acidified with 2 cc concen- 
trated HC1 and extracted once with 50 cc of ethyl ether in a 300 cc 
separatory funnel. The ether was washed in the same separator}' 
funnel with 5 cc NaOH (2.5%) and 5 cc dilute HC1. The ether was 
evaporated over 10 cc water and 5 cc of the water tested for indol by 
the method given in 5, B. In order to determine the percentage of 
recovery of indol in the first 100 cc of distillate, a culture tube inoc- 
ulated with an organism from the pink salmon was incubated for a 
week and then distilled. Ninety four per cent of the total amount re- 
covered was secured in the first 100 cc. These results indicated that 
it was not necessary to distill more than 100 cc. 

In the summary (table 16) the percentage of indol producers 
ranges from to 66. The lowest percentages are from bacteria from 

Table 16. Summary of indol production by bacteria from raw 
and canned salmon. 



Months 







since 


No. 


organisms 


Indol 


Skatol 




-Total 


xirce 


Species 


bacteria 




tested 


positive 


positive 




positive 






taken 


Nogr 


'th Growth 






No 


Per cent 




King 


13 


3 


40 


2 


1 


3 


7.2 




Red 


11 


10 


12 
















Coho 


11 


9 


8 
















Pink 


12 


7 


25 


7 





7 


28.0 




Chum 


10 


1 


20 


9 





9 


45.0 


2 


All 




6 


51 


9 





9 


17.6 


3 


All 


2 


4 


36 


16 


1 


17 


47 


4 


King 


1 




20 


10 





10 


50 


4 


Pink 


1 




31 


17 





17 


54 


4 


Red 


1 




24 


15 


1 


16 


66 


4 


Chum 


1 




9 


5 





5 


55 


5 


Chum 


1 




23 















Total 



299 



90 



93 



31 



1922 Clough; on Indol and Skatol in Salmon 255 

sources where they have been subjected to unfavorable environ- 
ment, such as low storage temperatures, long period of cultivation 
on artificial media, etc. The bacteria in source 5 are those hardy 
enough to survive cold storage temperatures for three months ; ap- 
parently all the indol producers were killed by this treatment. The 
bacteria from the red and coho salmon, source 1, had been on artificial 
media for 11 months and had been transferred only twice, while 
those from the other three species had been transferred three times. 
Several of the cultures were apparently dead, since they gave no 
growth in Dunham's media. The bacteria found in canned salmon 
were the survivors of a rather rigorous treatment. The spore-formers 
are of course the ones most likely to survive hardship, and it would 
seem that as a class they are not as likely to produce indol as the non- 
spore-formers. Even under the most favorable circumstances only 
66% of the bacteria taken from raw salmon produced indol, while 
some of those which did not form indol produced a putrid odor. It 
is therefore easily possible for bacteria to decompose salmon without 
the formation of indol or skatol. Regarding this, Ef front says: "We 
may conclude from the preceding that the presence of indol is not an 
infallible characteristic of putrefaction. Numerous aerobes and even 
certain anaerobes, like B. putrificus, which cause the disintegration of 
albumin to the most simple substances, do not, however, yield phenol 
or indol. The production of indol indicates merely a mode of attack, 
a particular direction given to the dismemberment. In other words, 
it corresponds only to the secretion of an amidase specific for this 
transformation." 

Skatol formation by bacteria was induced (table 16) by only 3 
of the bacteria studied in this investigation, when growing in Dun- 
ham's media, while 90 formed indol. It is possible that all those bac- 
teria forming indol might, under a different set of conditions, form 
skatol ; but it seems more probable that in those tests in which skatol 
was formed there was present a specific skatol-forming organism. 
Such an organism was isolated from culture No. 32 taken from raw- 
king salmon (source 1 )and was found to be identical with an organism 
isolated from canned macaroni. No description of this microorganism 
has been found in the literature, and it is probably a new species. It 
is a large, rod-shaped, motile bacterium which, on sporulation, becomes 
clostridium-shaped, with a large cylindrical spore. It is an obligate 
anaerobe but has been grown in paraffin-stratified broth in association 
with a facultative anaerobe from which it has been found rather dif- 
ficult to separate it. The facultative anaerobe is also rodshaped, mo- 



256 



Publ. Puget Sound Biol. Sta. 



Vol. 3, No. 67 



tile, gram-positive, and spore- forming; it is able to grow in oxygen 
tensions from almost zero to atmospheric. 

B. Formation of indol by scorching proteins 

Rohmann (1908) states that both indol and skatol may be formed 
from tryptophan by heat. Since salmon flesh carries tryptophan, it 
appeared that scorching might produce indol in perfectly fresh sal- 
mon. This had two important bearings on our problem ; first, the 
possibility of indol formation during the distillation in the indol deter- 
mination, and second, its possible formation during the commer- 
cial canning of fish. The first possibility is discussed here, and the 
second in 7, C. To eliminate the possibility of the formation of indol 
during its distillation from salmon, the distilling flask was never heat- 
ed over a free flame but in a bath of nearly saturated salt solution. 

To test the effect of scorching on salmon and other proteins, sev- 
eral experiments were carried out among which was the following 
one. Various substances containing protein were scorched in tin cans, 
transferred to flasks and distilled in the usual way. The distillates 
were extracted with ether, which was washed and evaporated. The 
water test solutions were divided into two parts, one of which was 
tested with Ehrlich's reagent, and the other with the Vanillin-HCl re- 
agent. The colors produced by the former reagent were extracted 
four times with chloroform and the residual color noted. The re- 
sults are given in tables 17 and 18. 

Table 17. Color produced by Ehrlich's reagent in the indol test on- 
various scorched foods. 



Substance 
scorched 

Peas, 

canned 

Beans, 
canned 

Eggs, 
fresh 

Salmon, 
canned 

Gelatin, 
Knox 



Initial color. 

Slight; like 
indol 

Strong ; purple ; 
like indol 

Very strong ; 
purple ; like 
indol 

Strong, purple ; 
like indol 



Strong ; more 
purple than 
the others 



1. 

Pink 

Pink 

Very- 
strong 
pink 

Pink 
with 
orange 

Slight 
orange 



Extractions with chloroform 
3. 4. 



2. 

Slight 

Pink 

Strong 
pink 



Very si. None 
Slight Very si. 
Pink Slight 



Residual color 
equivalent to 

1 mmg indol 
1 mmg indol 
3 mmg- indol 



Violet Pink Trace 1 mmg indol 



Slight 
violet 



Trace None 



Strong dark 
purple color ; 
over 20 mmg 
indol 



1922 C lough; on Indol and Skatol in Salmon 257 

Table; 18. Color produced by the vanillin-HCl reagent in the indol 
test on various scorched foods. 

Substance scorched Colors produced 

Peas, canned Weak color ; looks about right for indol 

Beans, canned Stronger color than produced in the case of the 

peas ; looks like the color produced by indol 

Eggs, fresh Very strong color; correct for indol 

Salmon, canned Strong color; correct for indol 

Gelatin, Knox Strong color; looks almost like the indol color. 

It appears that either indol, or a substance resembling it very 
closely, is formed by the scorching of those proteins which contain the 
tryptophan group. Gelatin does not contain this group, but a color is 
obtained from the scorched gelatin by both the Ehrlich and Vanillin 
tests which closely resembles the color produced from other scorched 
proteins which do contain the tryptophan group. However, since the 
color produced by the Ehrlich test did not extract with chloroform in 
the case of gelatin, but did in the case of all the other proteins tried, 
it appears that the color produced from gelatin was not due to indol. 

C. Formation of indol during the processing of salmon. 

Since indol apparently may be formed by scorching salmon, it 
seemed desirable to determine whether it may be formed during the 
commercial processing of salmon. This did not seem probable, but in 
order to be able to state definitely that indol found in cans was the 
result of bacterial action and not of the cooking process, it was nec- 
essary to prove or disprove the possibility of its formation during 
processing. 

A piece of fresh king salmon was obtained and ten half pound 
(227 g) cans filled. These were tightly closed and immediately 
placed in the pressure cooker, where they were cooked for 90 minutes 
at 240° C. The cans were then removed from the cooker and cooled. 
Eight cans were recooked and cooled as before. The same process 
was repeated on six cans, then on four, and finally on two. So that 
cans which had been cooked one, two, three, four and five times, were 
obtained. Five of the cans representing the five periods of cooking 
were opened, carefully examined, and the indol determined. The 
other five cans were later used in the determination of volatile nitro- 
gen. The raw fish used, when tested for indol, gave a negative test. 
The experiment was repeated, and the results are given in table 19. 



258 Publ. Puget Sound Biol. Sta. Vol. 3, No. 67 



The indol is expressed in mmg per 100 g of salmon. Ehrlich's re- 
agent used. 

Table) 19. Formation of indol during the processing of salmon. 
Experiment In raw fish 1st cook 2nd cook 3d cook 4th cook 5th cook 



1 


0.0 


0.3 


0.6 


0.8 


1.0 


1.3 


2 


0.1 


0.3 


0.6 


0.6 


1.1 


1.2 



A small amount of indol is apparently formed during the process- 
ing of salmon but the amount formed during the usual processing (1st 
cooking above) is so small as to be practically negligible. The amounts 
obtained from successive cookings show a gradual and regular increase. 
The odor varies from normal, through slightly scorched to strongly 
scorched, while at the same time the fish takes on a scorched flavor. 
The color grows gradually poorer and the texture softer. The indol 
color extracted well with chloroform in each case. 

D. Effect of exhaust on the indol content of canned salmon. 

Most of the canned salmon is exhausted before the cans are 
tightly closed and placed in the retorts. This is usually accomplished 
by passing the filled cans through a steam box either without the tops 
or with the tops loosely clinched on. During this process the contents 
of the can becomes heated, and a part of the air is expelled, resulting 
in a partial vacuum after the cans are tightly closed, cooked and 
cooled. The question arises as to the effect of this exhaust on the 
indol content and the odor of canned fish. Several experiments were 
carried out, using king, pink and chum salmon in various stages of 
decomposition. Each fish was cut into sections containing slightly 
more than one pound (454 g) each. These sections were divided into 
two equal pieces and each piece placed in a half pound (227 g) can. 
The cans were marked as usual to show the fish and section, and in 
addition the cans . from one side were marked "E" and those from the 
other side "N". The covers of the cans marked "E" were loosely 
clinched and the cans placed in steam at 100° C. (212° F.) for 12 
minutes, when the covers were tightly rolled on. The cans marked 
"N" were tightly closed while the cans were cold. All of the cans 
were cooked for 80 minutes at 115.5° C. (240° F.) 

After the cans had been stored for a few weeks they were ex- 
amined according to our usual method. The odor of the cans was 
noted very carefully by three to five men acting independently and 
without knowing which cans were exhausted and which were not. 



1922 C lough; on Indoi and Skatol in Salmon 259 

The odor of stale and tainted salmon is apparently slightly im- 
proved by exhausting the can; for when the separate results of the 
different examiners were compared it was found in nearly every case 
that the can in each pair which had been adjudged slightly better in 
odor was the exhausted can. The indol content in the two cans taken 
from the same section of the fish, while frequently markedly differ- 
ent, also showed this difference to be as often in favor of the ex- 
hausted can as of the unexhausted can. Averages for exhausted and 
unexhausted cans were nearly equal in most cases. Apparently, there- 
fore, exhausting the can has little effect on the indol content. 

8. Other decomposition changes 

An attempt was made to use some of the other decomposition 
changes and products as measures of decomposition. As stated be- 
fore, ammonia is without doubt formed from the amino acids dur- 
ing decomposition. However, there is good reason to believe that it 
may also be formed during the cooking process, creating a doubt as 
to the origin of the ammonia found in the can. Fatty acids may be 
formed progressively from fats during decomposition, but it is certain 
that they may also be formed by heat and pressure. Whether the 
cooking process in the case of salmon is severe enough to bring about 
this hydrolysis is a question to be settled by experiment. During the 
decomposition of salmon a peculiar substance is formed which pro- 
duces a strong biting sensation when placed on the tongue ; it also at- 
tacks the skin on the back of the hand. This substance is mentioned 
in 2. An attempt was made to isolate it. 

A. Volatile nitrogen as a measure of decomposition 

There are several methods for determining the volatile nitrogen 
based on distillation or on aeration. The methods used by Loomis 
(1912) employed distillation from an alkaline medium into standard 
acid, while Weber (1921) used a modification of the Folin aeration 
method. The latter method is given by the Assoc. Off. Agric. Chem- 
ists (1919) as tentative, and was selected, with certain modifications, 
for our use. On account of the lack of time, no work was done on 
the distillation methods. Leach (1920) recommends the use of al- 
cohol in the aerating cylinder, and this was found to reduce the froth- 
ing to some extent. The apparatus used consisted of six complete 
units, so that three determinations in duplicate could be made simul- 
taneously. The air was passed through under pressure, first being 



260 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

washed in a cylinder containing concentrated sulphuric acid. From 
the washing cylinder the air passed through six rubber tubes pro- 
vided with regulating pinch cocks, was conducted by glass tubes to 
the bottom of the aeration cylinders, and after bubbling up through 
the mixture of fish and chemicals, was passed through the Folin ab- 
sorption tube into N/50 H 2 S0 4 contained in a 250 cc graduated cyl- 
inder. The apparatus, as purchased, provided for the use of bottles 
four inches (10.2 cm) high for the absorption of the volatile nitro- 
gen compounds ; but experiments showed that a much smaller volume 
of air could be passed through these bottles than through the 250 cc 
cylinders, and consequently the time of aeration would need to be 
correspondingly increased. 

Twenty five grams of finely ground fish was placed in each 
aeration cylinder with 150 cc of water, 1 cc of saturated potassium 
oxalate solution, 25 cc of alcohol, a few drops of phenolphthalem and 
enough saturated potassium carbonate solution to render the mixture 
alkaline. Air was passed through as rapidly as possible for six hours 
and the acid in the absorption cylinder titrated against N/50 NaOH, 
using sodium alizarinsulphonate as an indicator. 

Experiments were now performed to determine whether there 
was an increase in volatile nitrogen during decompositkiii, ^nd also 
whether volatile nitrogenous compounds were formed during the cook- 
ing process. 

The increase in volatile nitrogen during decomposition was tested 
by using samples taken from the experimental packs of salmon in var- 
ious stages of deterioration as described in 6. Of course all of these 
samples were canned, so when comparing the results it is necessary to 
assume that if volatile nitrogenous compounds were formed in the 
canning process the same amount was formed in each of the cans 
used. The five species were used; the chemical results are given in 
table 20. 

"TabIvE; 20. Volatile nitrogen in salmon canned at different stages 
of decomposition. 



Hours out 




Volatile nitrogen mg 


per 100 g 


of fish 




of water 
when canned 














Pink 


Sockeye 


Chum 


King 


Coho 


Average 


24 


17.3 


22.3 


25.4 


34.0 


40.9 


28.0 


48 


21.6 


19.7 


31.7 


37.9 


38.5 


29.9 


72 


24.3 


24.6 


35.1 


33.8 


43.6 


32.7 


96 


32.3 


26.1 


29.7 


37.9 


48.4 


31.3 


120 


44.5 


41.4 


36.6 


46.3 


55.4 


44.8 


144 


54.3 


45.1 


47.1 


40.4 


55.3 


48.4 



1922 



Clough; on Indol and Skatol in Salmon 



261 



The results show a rather consistent increase in volatile nitrogen 
from day to day during decomposition in each of the five species. 
There are a few instances in which the amount decreases ; but it must 
be remembered that a different fish was used for each can, and that 
some of the fish examined on one day appeared to be in better con- 
dition than some of those which were examined and canned the day 
before. Furthermore, the volatile nitrogen formed during the cook- 
ing and during storage must be considered. For the purpose of cor- 
relation the amount of indol in these cans was also determined and 
is given in table 21. 



Tabids 21. Indol in salmon canned at different stages of decom- 
position. 



Hours out 
of water 
when canned 

24 

48 

72 

96 
120 
144 



45.0 



Indol mmg per 100 g of fish 



Pink Sockeye Chum 



Lost 



Kim 



Coho 



Average 



0.2 


0.2 


0.1 


0.1 


0.15 


1.0 


1.8 


0.8 


0.6 


1.05 


3.0 


4.5 


3.3 


1.3 


3.0 


10.0 


16.0 


20.0 


7.6 


13.4 


22.5 


52.0 


24.0 


15.7 


28.5 


50.0 


80.0 


22.0 


16.0 


42.6 



Both the indol and the volatile nitrogen increase ; but since the 
former starts from almost zero while the latter starts from an un- 
known quantity which depends upon the factors of cooking and stor- 
age, it is obvious that indol possesses advantages over volatile nitrogen 
as a measure of decomposition. 

The increase in volatile nitrogen during the cooking process was 
now investigated. Fresh salmon was obtained, the volatile nitrogen 
determined on a portion, and the rest placed in two half-pound (227 
g) cans, one of which was given the usual cooking (80 minutes at 
115.5° C, or 240° F.) and the other cooked twice. The volatile ni- 
trogen in each can was then determined. The determinations were 
made in duplicate. A great deal of difficulty was experienced in the 
determination of volatile nitrogen in raw salmon on account of ex- 
cessive frothing. Paraffin oil, kerosene, alcohol and other materials 
were used, as were various types of baffle plates, but the frothing 
continued. Although this experiment was repeated several times, only 
once could results be obtained with raw salmon on account of this 
frothing. These results are given in table 22. 



262 Publ. Puget Sound Biol. Sta. Vol,. 3, No. 67 

TabIv£ 22. Increase in volatile nitrogen during the canning 
process. 

Volatile nitrogen, 
Description of sample mg per 100 g 

Raw salmon 11.6 

Salmon cooked once 22.8 

Salmon cooked twice 34.0 

The results are remarkably uniform and show that cooking splits 
up the nitrogenous compounds and forms volatile alkaline substances. 

Some of the cans which had been packed for the determination 
of the amount of indol formed in salmon during the canning process 
(7, C) were used for the determination of volatile nitrogen. These 
cans had been cooked from one to five times the normal cooking 
process. Unfortunately no determination of the volatile nitrogen was 
made in the raw fish; but the indol determination was practically neg- 
ative, indicating that the fish used was in good condition. The re- 
sults are given in table 23. 

Table: 23. Increase in volatile nitrogen during repeated process- 
ing. 

Volatile nitrogen, 
Description of sample mg per 100 g 

Cooked once 45.1 

Cooked twice 45.7 

Cooked 3 times 56.8 

Cooked 4 times 57.9 

Cooked 5 times 64.2 

These results show a slight increase for each successive cooking, 
demonstrating that volatile nitrogenous compounds are split off by it. 
The amount found after the first cooking is much higher than given 
in table 22 ; this difference may be due to the fact that the samples 
used had been in the cans for nearly a year. Weber and Wilson 
(1919), when working on canned sardines, found the volatile nitro- 
genous compounds to increase during storage. This increase might 
explain why the increase from one cooking to another in table 23 was 
so much less than in table 22, since more ammonia might be formed 
during storage in those cans which had the smallest amount at the be- 
ginning of the storage period. 

The results of both experiments show that the cooking process 
does increase the volatile nitrogenous constituents of salmon. The 



1922 Clough; on Indol and Skatol in Salmon 263 

results also tend to confirm the report of Bidault and Couturier 
(1920) that the amount of ammonia in canned meat is a function of 
the heat of sterilization. 

B. . Increase in free fatty acids as a measure of decomposition in 

salmon. 

Weber (1921), in his work on the Maine sardine, determined 
the fat and the free fatty acids in sardines which had been held in 
brine for periods ranging from 2 to 96 hours, and concluded that no 
change of a significant nature was shown by the results. However, 
we made a few experiments, none of which gave satisfactory results. 

The first method used was based on that of Folin and Wentworth 
(1910) for the determination of fat and fatty acids in feces. The 
cans of salmon used were opened, carefully examined, and then thor- 
oughly mixed. Ten gram portions were weighed out on lead dishes 
and dried in a vacuum oven at 80° C, cooled and weighed. The lead 
dishes containing the dried salmon were then placed in Soxhlet ex- 
tractors and extracted for 16 hours with anhydrous ether containing 
sufficient anhydrous HC1 to make the ether solution approximately 
tenth normal. The ether was distilled from the flasks z~d petroleum 
ether added. After standing over night the petroleum ether solution 
was filtered into a weighed flask and the residue washed with petrol- 
eum ether. The petroleum ether was then evaporated ; the residue 
weighed, dissolved in benzene, and titrated with -N/10 sodium ethylate, 
using phenolphthal.ein as an indicator. 

This method did not prove satisfactory and the experiments were 
repeated using anhydrous ethyl ether instead of the ether-HCl solvent. 

In the investigation of the free fatty acid in salmon canned at 
different stages of decomposition, the cans used were part of the ex- 
perimental pack of king salmon described in 6, B. Two portions of 
each were taken from each can, one portion (a) was extracted with 
the ether-HCl solvent and the other (b) with ether alone. The re- 
sults are expressed as milligrams of stearic acid per gram of fat in 
table 24. 

The results for solids are uniformly higher in the (b) samples 
than in the (a) samples; this is due to the fact that the (b) samples 
were secured after the ground fish had been standing exposed to the 
air for an hour and had apparently lost some of the moisture. The 
fat is also higher in most of the (b) samples. The results for fatty 
acids are very contradictory. The results for the (a) samples are 
higher than those for the (b) samples, except in the case of K2, and 



264 Publ. Puget Sound Biol. Sta. Voh. 3, No. 67 

Tabids 24. Solids, fats, and fatty acids, in king salmon canned at dif- 
ferent stages of decomposition. 



Hours out of 






Fatty acids as 


water before 


Solids, 


Fat, 


stearic acid 


canning 


per cent 


per cent 


mg per g fat 


24 


(a) 39.70 


17.00 


114.2 




(6) 39.89 


17.25 


74.0 


48 


(a) 39.96 


14.63 


102.9 




(b) 40.41 


16.92 


37.7 


72 


(a) 44.65 


24.50 


135.5 




(b) 45.12 


21.15 


70.0 


96 


(a) 39.56 


17.16 


93.7 




(b) 40.24 


17.81 


113.2 


120 


(a) 38.67 


15.55 


.95.2 




(6) 39.90 


16.32 


70.4 


144 


(a) 38.67 


13.74 


115.2 




(&) 39.44 


14.22 


96.9 



Can No. 
A2 
D2 
G2 
K2 
N2 
P2 



suggest two possibilities ; either the acid in the ether-HCl solvent par- 
tially hydrolyzed the fat, or else the HCl was not entirely removed 
when the ether was evaporated. Even the (b) samples, with which 
no HCl was used, do not show a consistent increase during decompo- 
sition. The method appears to be of little value as a measure of de- 
composition in canned salmon. 

In the investigation of the free fatty acid in salmon before 
and after canning, portions of a fresh king salmon were used. The 
solids, fats and fatty acid were determined both before and after 
canning. Anhydrous ether was used for extraction. The results 
are given in table 25. 

Tabi^ 25. Free fatty acid in salmon before and after canning. 

Description of sample 

Raw salmon . ■__. 

Canned salmon 

The results are not favorable to the theory that the fatty acid is 







Fatty acids as 


Solids, 


Fat, 


stearic acid 


per cent 


per cent 


mg per g fat 


25.64 


5.36 


47.6 


25.64 


5.31 


23.8 


31.95 


9.18 


13.9 


31.94 


9.11 


14.0 



1922 Clough; on Indol and Skatol in Salmon 265 

increased during the canning process, but the data is of course too 
meager to warrant the drawing of definite conclusions. 

The amount of free fatty acid was also determined in some king 
salmon which had been cooked one, three and five times. These 
cans were a part of those described in 7, C. The results are given 
in table 26. 

Tabi,3 26. Effect of cooking on the free fatty acid content of canned 

salmon 

Fatty acids as 
Description of sample Fat, stearic acid 

per cent mg per g fat 

Cooked once 7.94 173.5 

Cooked 3 times 8.24 170.5 

Cooked 5 times 6.45 205.8 

The results do not show a satisfactory correlation between the 
number of times cooked and the amount of free fatty acid. 

The three experiments described above indicate that the deter- 
mination of the free fatty acid is not likely to be of value in detect- 
ing decomposition in canned salmon. 

C. Formation of a substance having a biting taste. 

Tainted canned salmon, when placed on the tip of the tongue, 
produces a sensation similar to that produced by "strong" cheese. 
If the salmon be rubbed on the back of the hand the skin becomes 
irritated and itches. Canned salmon in good condition does not 
produce these sensations. Some new substance has been formed 
during the decomposition. The same substance, or at least one 
giving the above sensations, is found in partially decomposed tuna. 
In both cases its presence appears to be associated with "honey- 
combing." Several attempts were made to isolate the substance; 
but although it was obtained in a highly concentrated form, as judged 
by the effect on the tongue, it was never obtained in a pure state. 

9. Summary 

The canning of salmon constitutes one of the most important 
industries of the Pacific coast of North America. Salmon, in com- 
mon with other fish, are delicate, easily injured and very easily 
decomposed ; and there are many opportunities for spoilage between 



266 Publ. Puget Sound Biol. Sta. Voi,. 3, No. 67 

the time they are taken from the water and the time they are 
canned. A systematic method for the examination of canned salmon 
has long been needed, and such a method is herein outlined. 

The literature on the chemical composition of fish flesh is 
reviewed. Original data are given, with food value, based on the 
analysis of 643 cans of salmon comprising individual fish of the 
five species from each important canning district. The decomposi- 
tion of fish flesh together with the utilization of certain decomposi- 
tion products as a means of estimating the amount of spoilage is 
discussed. Indol and skatol are the products finally selected as the 
most suitable for this purpose. 

The principal color tests for indol and skatol are given: the 
Ehrlich, Herter and dimethylaniline tests are chosen as the most 
sensitive. These three tests are modified and improved and their 
delicacy increased; a method for the determination of indol and 
skatol in salmon is developed. 

A biochemical study is made of the five species of salmon, 
covering the physical and bateriological changes, and the appear- 
ance and increase of indol and skatol during progessive decomposi- 
tion. Data regarding the 138 salmon studied along each of the 
above lines of investigation are given in tables showing the average 
daily decomposition changes, and in curves showing the increase in 
indol. Skatol was not found in any of the raw salmon, or in the 
experimental cans prepared from them; but was found in several 
commercial cans of salmon. An organism capable of forming skatol 
in salmon flesh was, however, isolated from one of the raw king 
salmon. Indol was found in small amounts in each of the salmon 
examined at the end of 48 hours storage, but only three of them con- 
tained more than 1.5 mmg per 100 grams. In general it may be 
said that when this quantity of indol is found in canned salmon a 
considerable degree of decomposition has taken place. 

Indol was quantitatively determined in 544 commercial cans of 
salmon. As some of these cans had a strong tainted odor and yet 
contained very little indol, the absence of indol cannot be taken as 
complete evidence that decomposition has not taken place. The 
experimental or laboratory packs of salmon contained more indol 
than commercial packs; all experimental cans classed as tainted 
contained more than 1.5 micromilligrams per 100 grams. 

Rather close correlation was found between the number of 
bacteria present from day to day during spoilage and the indol 
content; this is shown by means of curves. A close correlation was 



1922 Clough; on Indol and Skatol in Salmon 267 

also found between the storage temperature and the indol content ; 
king salmon stored at the highest temperature had the most indol, 
while the sockeye and coho salmon stored at the lowest temperature 
had the least. 

The gills were found to have more bacteria and more indol than 
either the viscera or flesh. The flesh was sterile at the end of 24 
hours in most of the fish examined, and contained little or no indol. 
The cooked flesh appeared to contain slightly more indol than the 
raw, but this may be due to the action of the cooking process in 
bteaking down the cellular structure of the fish, resulting in a more 
rapid and complete liberation and distillation of the indol. The first 
cue of the fish, just behind the gills, was found to contain more indol 
than a cut through the middle of the fish in the region of the dorsal 
fin. 

Exhausting the cans of stale and tainted fish was found to 
improve slightly their odor, but there was apparently no change in 
the indol content. Indol, or a substance very closely resembling it, 
is apparently formed by the scorching of those proteins, including 
salmon flesh, which contain the tryptophan group. A small amount 
of indol may be formed during the processing of salmon, but this 
amount is so small as to be negligible. Excessive cooking produces 
slightly more indol, a scorched odor and flavor, and a slightly softer 
texture. 

The indol producing power of 299 different cultures of bacteria 
taken from raw or canned salmon was tested and only 31 per cent 
gave positive tests. The comparatively small number of these bacteria 
which produced indol. suggests a reason why so large a percentage of 
the tainted commercial cans contained little or no indol. 

The canning process appears to increase the amount of volatile 
nitrogen (ammonia and amines) in salmon. The volatile nitrogen 
content of salmon increases from day to day during decomposition 
with considerable regularity; but owing to the probability of its 
formation during the cooking process, it is not suitable as a measure 
of decomposition in canned salmon, although no doubt it is of value 
as a criterion in the case of raw salmon. 

Very unsatisfactory results were obtained in attempting to use 
the free fatty acids of salmon oils as a measure of decomposition 
of the fish from which they were extracted. Tainted canned salmon, 
when placed on the tip of the tongue, produces a sensation similar 
to that produced by "strong" cheese. A substance producing this 
sensation was obtained in a highlv concentrated but impure form, 



268 Publ. Puget Sound Biol. Sta. Voiy. 3, No. 67 

and the attempt to identify it was abandoned on account of lack of 
time. 

In conclusion it may be said that although the determination of 
indol cannot supplant odor and physical appearance in the examina- 
tion of canned salmon, it is nevertheless of value and affords con- 
siderable information as to the previous history of the sample. 

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